In contrast to eukaryotic aspartic proteases, bacterial aspartic proteases are generally located in the cytoplasm. We have identified a surface-associated mycobacterial aspartic protease, PecA, which cleaves itself and many other type VII secretion substrates of the PE_PGRS family. PecA is present in most pathogenic mycobacterial species, including M. tuberculosis. In addition, pathogenicity of M. marinum is reduced in the ΔpecA mutant, indicating that PecA contributes to virulence.Aspartic proteases are common in eukaryotes and retroviruses but are relatively rare among bacteria  this PE domain at the cell surface. Nearly all of the observed PE_PGRS proteins are processed by PecA. Interestingly, the protease itself is also a secreted PE protein and subject to self-cleavage. Furthermore, a defect in surface processing has no effect on the activity of the PE lipase protein LipY but does seem to affect the functioning of other virulence factors, as a pecA mutant strain of Mycobacterium marinum shows moderate attenuation in zebrafish larvae. In conclusion, our results reveal the presence of a functional aspartic acid protease in M. marinum that cleaves LipY, itself as well as other members of the PE_PGRS family. Finally, mutants lacking PecA show growth attenuation in vivo, suggesting that PecA plays a role during infection.Tuberculosis, one of the world’s most severe infectious diseases, is caused by  Mycobacterium tuberculosis, a facultative intracellular pathogen with a highly unique diderm cell envelope. This unique cell envelope is highly impermeable and requires specialized secretion systems for protein transport to the cell surface , respectively, which includes the Pro-Glu or Pro-Pro-Glu motifs close to the N terminus after which they were named . Althougonly), while others have C-terminal extensions of variable size. The largest subfamily of PE proteins in M. tuberculosis contains a glycine-rich C-terminal domain encoded by so-called polymorphic GC-rich repetitive sequences (PGRS) and are therefore known as PE_PGRS proteins. These proteins have been linked to immune modulation (–Some PE proteins contain only a PE domain (PEdulation –11. AlthM. tuberculosis (LipYtub). LipYtub functions as a surface-exposed triacylglycerol lipase. In M. tuberculosis, LipY is mainly expressed under hypoxic conditions and could be involved in lipid body formation  of M. tuberculosis. Somewhat weaker similarity (43% identity) is observed with the C-terminal domain of PE_PGRS16. Interestingly, the structure of the C-terminal domain of PE_PGRS16 has been solved and belongs to the family of aspartic proteases  and a model of MMARE11_28540 (amino acids 269 to 551 out of 554). Aspartic acids within the active site containing DTG/DSG motifs are shown in orange. (B) Sequence alignment performed by MUSCLE of putative aspartic proteases (domains) in M. tuberculosis, M. marinum, human pepsinogen A3 (PGA3), and HIV protease (HIV-PR). Boldface indicates identified protease that affects LipYtub processing. Underscoring indicates that aspartic acid 293 is mutated to glycine when referring to the catalytic dead mutant D293G. Red indicates the aspartic acid signature DTG/DSG motifs. The HIV protease sequence is repeated to simulate the two lobes found in the other proteases. Download FIG S1, PDF file, 1.9 MB.Comparison of the aspartic protease domains. (A) Structural comparison of the protease domains of PE_PGRS16 and MMARE11_28540. Side and front views depicting the crystal structure of protease domains of PE_PGRS16 . We expressed LipYtub with a C-terminal hemagglutinin (HA) tag in M. marinum wild type and the Δmmar_2933 strain. Western blot analysis showed that deletion of mmar_2933 completely abolished the processing of LipYtub  resulted in a slightly larger but still cleaved product of around 33 kDa . In. Intub. A150D from the Genapol supernatant (GS)   and perfce AAQAI . This cl10.1128/mBio.01951-19.2FIG S2M. marinum M wild type and wild type expressing A150DpSMT3-lipY were stained with Coomassie brilliant blue R250. The arrow indicates the protein band that was subjected to Edman sequencing. Download FIG S2, PDF file, 0.3 MB.Processing of LipY. Genapol supernatant fractions of Copyright © 2019 Burggraaf et al.2019Burggraaf et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the tub and LipYA150D takes place in the linker domain. However, LipY-OVA2, whose secretion was affected in mmarE11_28540::tr, does not contain this linker domain. This hinted at the possibility that MMAR_2933 also cleaves at a third location, inside the PE domain of LipY-OVA2. To explore cleavage inside the PE domain, we expressed LipY without the linker domain (LipYΔ98–201) in wild-type and Δmmar_2933 strains. In the wild-type strain, we observed both the full-length product of LipYΔ98–201 at 36 kDa and a processed fragment with an apparent weight of 25 kDa  corresponds to the size of the PE domain. These experiments demonstrate that MMAR_2933 is a protease that cleaves PE proteins, and therefore, we would like to rename this protein PE cleavage protein A (PecA).Cleavage of both LipYf 25 kDa . Similartub was analyzed by flow cytometry. This experiment showed that cell surface exposures of LipYtub were similar for the wild-type and the ΔpecA mutant strains  (tub by PecA does not affect LipYtub lipase activity in M. marinum.Previous research has shown that the presence of the PE domain reduces the lipase activity of recombinant LipYhia coli . To test LipYtub . Furtheron (POX) , demonst10.1128/mBio.01951-19.3FIG S3M. marinum wild-type and ΔpecA strains with or without expression of LipYtub. Lipase activity was measured by a DGGR assay. No lipase activity was detected following treatment with lipase inhibitor POX. Data are presented as mean ± SD values (n = 3). Download FIG S3, PDF file, 0.1 MB.Lipase activity of Copyright © 2019 Burggraaf et al.2019Burggraaf et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the tub: a PE domain followed by a linker domain and a C-terminal domain with enzymatic activity. To investigate whether PecA has the capacity to cleave itself, PecA with a C-terminal Myc tag was introduced in the M. marinumΔpecA strain. Western blot analysis showed that PecA had an observed molecular weight of approximately 42 kDa, instead of the expected 53 kDa, suggesting the removal of the 11-kDa PE domain (PecA is a PE_PGRS protein and has a similar domain structure as LipYE domain .D293G). This mutated PecA was not cleaved when expressed in the ΔpecA strain, demonstrating that the aspartic protease activity of PecA is indeed required for PecA self-cleavage  residues stood out . In addition, proximal to the secretion motif is a conserved glycine alanine motif at position 84. The presence of consecutive small amino acids within the PE domain combined with the approximately 11-kDa size of the cleaved N-terminal domain led us to hypothesize that the PE domain of PecA and LipYtub is cleaved near or inside the secretion signal motif  and studied its processing in M. marinum. The PecAA84D and PecAAA88ED mutants were both partially processed as wild-type PecA . The arrow indicates the fully cleaved form of PecA. PecA was detected with an anti-Myc antibody. Download FIG S4, PDF file, 0.4 MB.Processing of the PE domain. Immunoblot analysis of Copyright © 2019 Burggraaf et al.2019Burggraaf et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the tub and PecA, we hypothesized that PecA could also cleave other PE_PGRS proteins. We tested this hypothesis by immunoblotting lysates of wild-type and ΔpecA strains using a monoclonal antibody directed against the PGRS domain, recognizing various PE_PGRS proteins  with high sequence identity  and conserved synteny. The identity is especially conserved in the protease domain, implying a similar function. To investigate whether PE_PGRS35 processes PE_PGRS proteins in a similar fashion, we expressed this gene in M. marinumΔpecA and visualized the PE_PGRS proteins by immunoblotting. We observed full complementation, based on the identical PE_PGRS patterns between the complemented strain and the wild-type M. marinum strain  Growth curves of Mycobacterium marinum M strains. Wild-type, ΔpecA, ΔpecA plus mycpecA, or ΔpecA plus mycΔpecAD293G strains were grown in triplicate in 7H9 supplemented with ADC. OD was measured at indicated time points and plotted against time. (B and C) A. castellanii cells were infected with mEosFP-expressing wild-type and ΔpecAM. marinum M strains. Number of infected cells was determined at 4 and 24 hours postinfection by flow cytometry . (D) RAW 264.7 cells were infected with wild-type and ΔpecAM. marinum M strains containing mEosFP. The number of infected cells was determined 0, 20, and 43 hours postinfection by flow cytometry. Data are presented as mean ± SD values (n = 3) (10.000 cells per sample). Download FIG S5, PDF file, 0.5 MB.Effect of PecA on intracellular replication following infection in Copyright © 2019 Burggraaf et al.2019Burggraaf et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the M. marinum wild-type and ΔpecA strains. In contrast to the experiments with phagocytic cells, the ΔpecA strain showed reduced outgrowth (5-fold change) after 4 days compared to wild-type bacteria, when measured by fluorescence microscopy , ΔpecA (4 CFU), or ΔpecA plus mycpecA (9 CFU) M. marinum M strains. Graphs show absolute infection as counted by CFU per larva at 4 days postinfection. Data are presented as mean ± SD, and the 1-way ANOVA was used for statistical analysis on the log-transformed CFU data. The P value did not reach statistical significance when comparing CFU counts between the ΔpecA and ΔpecA plus mycpecA strains (P = 0.0944). Download FIG S6, PDF file, 0.4 MB.Deletion of Copyright © 2019 Burggraaf et al.2019Burggraaf et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01951-19.8TABLE S2Table S2, PDF file, 0.1 MB.Input CFU and number of larvae per experiment per strain. One nl was injected into the larvae. Pre, number of bacteria present in inoculum per nanoliter prior to injecting larvae. Post, number of bacteria present in inoculum per nanoliter after injecting larvae. n.d., not done; strains were not yet generated. Download Copyright © 2019 Burggraaf et al.2019Burggraaf et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the M. marinum-secreted PE_PGRS proteins by PecA, a PE_PGRS protein with aspartic protease activity  and PE_PGRS35 . All three proteins contain the DTG/DSG sequences that are characteristic for the catalytic domain of aspartic proteases. Whereas this previous study was unable to demonstrate proteolytic activity for PE_PGRS16, in our study we conclusively show that PecA is an active protease that is essential to process PE_PGRS proteins at the cell surface.Previously, Barathy and Suguna  resolvedtub, one of the PecA substrates, contains at least three different processing sites, two in the linker domain and one near the end of the PE domain. This extensive processing could be an exception, since we observe an apparent molecular weight difference consistent with the removal of the PE domain (or part thereof) for PecA and other PE_PGRS proteins. Mutations near or inside the secretion signal motif YxxxD/E of PecA resulted in reduced and even altered processing of the PE domain, demonstrating that the PE domain is cleaved by PecA. We were, however, unable to create a mutant that fully blocked processing by PecA. This is not unexpected, since cleavage sites of aspartic proteases, such as HIV-1 protease, are known to be difficult to predict. The HIV-1 protease cleavage sites not only rely on sequence but also are determined by local structure and physicochemical features of the cleavage site , as Abdallah et al. demonstrated by mass spectrometry (MS) analysis , and the exact role of each PE_PGRS proteins during infection is unknown. Previously, we have demonstrated that PE_PGRS proteins reduce virulence, since strains lacking PE_PGRS (and PPE-MPTR) secretion, exhibited increased virulence  redundancy with the two homologs, i.e., PE26 and PE_PGRS16.PecA from in vivo, the importance of PecA in pathogenic mycobacteria can be illustrated by its high degree of conservation in the mycobacterial genome. Most PE_PGRS proteins show high variability between clinical M. tuberculosis strains and accumulate mutations faster than expected  at 37°C.inum E11  and M (3inum E11  were groEscherichia coli DH5α and E. coli BL21 were grown in LB medium (Difco-BD Biosciences) at 37°C with shaking at 200 rpm.For all cultures, hygromycin was used at a concentration of 50 μg/ml, kanamycin was used at 25 μg/ml, and streptomycin was used at 30 μg/ml.M. marinumΔpecA was constructed using the mycobacteriophage method developed by the Jacobs laboratory . This construct was identified by screening for enhanced secretion after random mutagenesis of LipYtub-1–205 fused to chick ovalbumin (amino acids 259 to 357) in the pSMT3 plasmid. This work is described in the work of Burggraaf et al.  was amplified by PCR using primers PecA-pMV-Fw and PecA-pMV-Rv (primers are listed in tub was created by restriction cloning. PecAtub was PCR amplified from genomic H37Rv DNA with the primers Rv1983-Fw and Rv1983-Rv. The PCR product was digested with EcoRI and XhoI and ligated into the pST5552 plasmid (a pMV261-based vector with a theophylline-inducible riboswitch) digested with EcoRI and SalI. The plasmids were verified with sequencing.To create pMV361in vitro transcription, pET28A-iCAP was restricted with XhoI and NcoI. pecA and D293GpecA were amplified by PCR from pMV-pecA and pMV-D293GpecA, respectively, using primers PecA-pET-Fw and PecA-pET-Rv and introduced in pET28A (NcoI/XhoI) by infusion cloning.To enable M. marinum by electroporation and to E. coli DH5α by transformation according to standard heat shock protocol.Plasmids were introduced to M. marinum E11 transformed with pSMT3-2lipY-OVA  placed on a 7H10 plate supplemented with Middlebrook OADC (Difco-BD Biosciences). Plates were incubated at 30°C. When colonies formed, the membrane was transferred onto a new membrane already placed on a fresh plate and incubated overnight at 30°C. The second membrane was incubated with antibodies directed against the influenza virus hemagglutinin epitope  and the secondary antibody horseradish peroxidase-conjugated goat anti-mouse IgG . Signal was detected with enhanced chemiluminescence (ECL) Western blotting detection reagent (Amersham Bioscience). Selected transposon mutants and complemented strains were grown to an optical density at 600 nm (OD600) of 0.8 to 1.2 in 7H9 supplemented with Middlebrook ADC (Difco Biosciences), 10-μl drops were placed on the nitrocellulose filter on a 7H10 plate, and the procedure for analyzing secretion was repeated.After transposon mutagenesis, pecA and pET28A-D293GpecA were expressed with the PURExpress in vitro protein synthesis kit (New England Biolabs [NEB]) according to the manufacturer’s protocol. Briefly, 100 ng plasmid DNA was added to a mixture of solutions A and B, supplemented with RNase inhibitor (Thermo Scientific). The reaction mixture was incubated for 2 h at 37°C and subsequently put on ice for 15 min. Solubilization/denaturation (SD) buffer (containing 100 mM dithiothreitol and 2% SDS) was added before loading samples on the gel for SDS-PAGE.pET28A-600 of 0.8 to 1.2. Cultures were harvested, and the supernatant was filtered through an 0.2-μm Millipore filter and precipitated with 10% trichloric acetic acid (Sigma) and 36 μg/ml tRNA (Roche). Pellets were washed with phosphate-buffered saline (PBS). Two OD units was taken and dissolved in SD buffer to a concentration of 0.02 OD/μl, making the pellet sample. Two OD units was incubated with 0.5% Genapol X-080 (in PBS) for 30 min at room temperature (RT). After spinning down, the supernatant was taken and diluted with SD buffer to a final concentration of 0.016 OD/μl, forming the Genapol supernatant fraction. The remaining pellet was washed with PBS and dissolved in SD buffer to a concentration of 0.02 OD/μl, forming the Genapol pellet fraction. Pellet samples were sonicated. All samples were incubated 5 min at 98°C.One day prior to making samples, cultures were washed with 7H9-0.2% dextrose-0.2% glycerol-0.05% Tween 80 to remove ADC. Cultures were grown in this medium at 30°C, with shaking at 90 rpm to an ODSamples were loaded on a 12.5% SDS-PAGE gel. Subsequently, proteins were transferred to nitrocellulose membranes for Western blotting. Blots were blocked with 5% skimmed milk in PBS and incubated with antibodies recognizing the influenza virus hemagglutinin tag , GroEL2 , PGRS domain , or the 600 of 0.8 to 1.2 in 7H9 with ADC and without Tween 80. Bacteria were pelleted, washed with PBS with 0.5% bovine serum albumin , and incubated for 1 h with antibodies recognizing the influenza virus hemagglutinin tag . After washing with PBS with 0.5% BSA, bacteria were incubated with secondary goat anti-mouse IgG conjugated to Alexa 647 antibodies for 1 h. After washing with PBS with 0.5% BSA, bacteria were analyzed by flow cytometry . As a control, bacteria were incubated only with the secondary antibodies.Bacteria were grown to an ODt test. The fluorescent intensity data obtained from the zebrafish experiments were normalized and log10 transformed to obtain a Gaussian distribution. Normalization was performed by subtracting the geometric mean of uninfected larvae and setting the maximum value for each experiment arbitrarily to 10,000. Larvae with a negative fluorescence were set to 1 to allow for log10 transformation. A 1-way analysis of variance (ANOVA) was performed on the transformed data and on the CFU data to test the null hypothesis: there is no difference between the groups and equality between means. When statistical significance (P < 0.05) is reached, the P value is specified.Data obtained with the lipase assay were analyzed with an unpaired 4EHC) as a template  and 29 μg/ml tRNA. Samples were incubated 1 h on ice, before being washed with acetone. The supernatant was discarded, and pellets were dried at RT. Pellets were dissolved in SD buffer and incubated for 5 min at 98°C.Bacterial cultures were washed with 7H9-0.2% dextrose-0.2% glycerol and grown to an ODA150D was excised, and the 5 N-terminal amino acids were analyzed by Edman N-terminal sequencing .Samples were run on a 12.5% SDS-PAGE gel supplemented with 0.4 mM gluconic acid (Merck). The gel was blotted on a polyvinylidene difluoride (PVDF) membrane (Bio-Rad). The blot was stained with 0.1% Coomassie brilliant blue R250, 50% methanol, 1% acetic acid, and destained with 50% methanol. The band corresponding to the cleaved product of LipY600 of 0.2. One hundred sixty microliters of bacterial suspension was mixed with 25 μl development solution DGGR . Fluorescence was measured in a plate reader at an excitation of 530 nm and emission of 600 nm. Paraoxon  is a lipase inhibitor, used as a control for inhibition of LipY lipase activity.Bacteria were grown to mid-log phase in 7H9 medium (Difco) supplemented with 10% ADC (BD Bioscience) with 0.05% Tween 80 and diluted to an OD600 of 0.7 to 1). Bacteria were spun down at low speed for 1 min to remove the largest clumps, washed with 0.3% Tween 80 in phosphate-buffered saline (PBS), and sonicated briefly for declumping. Bacteria were resuspended in PBS with 20% glycerol and 2% polyvinylpyrrolidone and stored at −80°C. Before use, bacteria were resuspended in PBS containing 0.17% (vol/vol) phenol red (Sigma) to aid visualization of the injection process. To determine the exact number of bacteria injected, the injection volume was plated on 7H10 plates containing the proper antibiotic selection. At 4 days postinfection (dpi), larvae were analyzed with an Olympus IX83 fluorescence microscope. Bright-field and fluorescence images were acquired with an Olympus IX83 microscope equipped with an Orca-flash 4.0 LT camera. Infection levels were quantified with CellProfiler 3.15. Zebrafish were manually delineated followed by integrating the fluorescent intensity of the entire fish.Transparent casper zebrafish larvae were removed from their chorion with tweezers and infected at 1 day postfertilization (dpf) via the caudal vein with a bacterial suspension. The injected CFU was determined before and after injection of the larvae to rule out blockage of the needle , on Middlebrook 7H10 agar.


Staphylococcus aureus is regulated via autoinducing peptide (AIP)-dependent activation of the sensor kinase AgrC, which forms an integral part of the agr quorum sensing system. In response to bound AIP, the cytoplasmic domain of AgrC (AgrC-cyt) undergoes conformational changes resulting in dimerization, autophosphorylation, and phosphotransfer to the response regulator AgrA. Naturally occurring mutations in AgrC-cyt are consistent with repositioning of key functional domains, impairing dimerization and restricting access to the ATP-binding pocket. Strains harboring specific AgrC-cyt mutations exhibit reduced AIP autoinduction efficiency and a timing-dependent attenuation of cytotoxicity which may confer a survival advantage during established infection by promoting colonization while restricting unnecessary overproduction of exotoxins.Virulence factor expression in  Staphylococcus aureusagr locus responsible for quorum sensing (QS)-dependent virulence gene regulation occur frequently during host adaptation. In two genomically closely related S. aureus clinical isolates exhibiting marked differences in Panton-Valentine leukocidin production, a mutation conferring an N267I substitution was identified in the cytoplasmic domain of the QS sensor kinase, AgrC. This natural mutation delayed the onset and accumulation of autoinducing peptide (AIP) and showed reduced responsiveness to exogenous AIPs. Other S. aureus strains harboring naturally occurring AgrC cytoplasmic domain mutations were identified, including T247I, I311T, A343T, L245S, and F264C. These mutations were associated with reduced cytotoxicity, delayed/reduced AIP production, and impaired sensitivity to exogenous AIP. Molecular dynamics simulations were used to model the AgrC cytoplasmic domain conformational changes arising. Although mutations were localized in different parts of the C-terminal domain, their impact on molecular structure was manifested by twisting of the leading helical hairpin α1-α2, accompanied by repositioning of the H-box and G-box, along with closure of the flexible loop connecting the two and occlusion of the ATP-binding site. Such conformational rearrangements of key functional subdomains in these mutants highlight the cooperative response of molecular structure involving dimerization and ATP binding and phosphorylation, as well as the binding site for the downstream response element AgrA. These appear to increase the threshold for agr activation via AIP-dependent autoinduction, thus reducing virulence and maintaining S. aureus in an agr-downregulated “colonization” mode.Mutations in the polymorphic IMPORTANCE Virulence factor expression in Staphylococcus aureus is regulated via autoinducing peptide (AIP)-dependent activation of the sensor kinase AgrC, which forms an integral part of the agr quorum sensing system. In response to bound AIP, the cytoplasmic domain of AgrC (AgrC-cyt) undergoes conformational changes resulting in dimerization, autophosphorylation, and phosphotransfer to the response regulator AgrA. Naturally occurring mutations in AgrC-cyt are consistent with repositioning of key functional domains, impairing dimerization and restricting access to the ATP-binding pocket. Strains harboring specific AgrC-cyt mutations exhibit reduced AIP autoinduction efficiency and a timing-dependent attenuation of cytotoxicity which may confer a survival advantage during established infection by promoting colonization while restricting unnecessary overproduction of exotoxins. Staphylococcus aureus relies on a finely balanced network of transcriptional and translational regulators. These systems integrate the response to a wide variety of stimuli, including pH, oxidative stress, temperature, cell wall damage, nutrient availability, and cell population density (–agr) is a cell-cell communication or “quorum sensing” (QS) system integral to the staphylococcal virulence factor regulatory network. It responds to bacterial cell population density by sensing a threshold concentration of the cognate QS signal molecule. This leads to the upregulation of diverse exotoxin genes  while downregulating cell wall proteins responsible for, e.g., surface attachment and biofilm formation , controlled by the agrP2 and agrP3 promoters, respectively  ST22 lineage strain harboring a single AgrC Y223C cytoplasmic domain substitution  and AIP production, focusing on the dynamics of agr activation and their impact on the conformation of the AgrC cytoplasmic domain using molecular dynamics simulations. The findings presented have potential implications for the adaptation of S. aureus during infection and the clinical potential of agr antagonists as antivirulence agents.The divergent nature of the and agrC , 11. Foraptation –15 genertitution  that swiS. aureus ST22 methicillin-sensitive (MSSA) strains isolated from clinical samples, we noted a subset of high-level PVL producers on immunoblotting, with one exception: strain TS13, a low-level PVL producer . To determine the relative expression levels of the PVL genes in TS13 and TS14, quantitative reverse transcription-PCR (RT-PCR) was carried out using lukF-PV and found to be 59.7-fold higher  in TS14  between the two strains AIP-1 when added at 0 or 2 h after inoculation but not after 4 or 6 h of growth  , TS13 and TS14 contain a shared SNP resulting in a Y-to-F substitution at residue 251 (Y251F)   . The dif (Y251F) . A SNP w (N267I) . An SNP nd SigB 13. No SNPagrC was carried out on the agr group 1 bioreporter ROJ143 that produces light in response to exogenous AIP-1 but does not produce endogenous AIPs (50) of 9.6 ± 1.6 nM for the AgrC-N267I bioreporter compared to 3.4 ± 0.6 nM for the control (P < 0.01)   showed a marked delay in agrP3 expression over the growth curve that could be restored to the wild-type (MY15) profile by exogenous AIP-1 and was completely inhibited by (Ala5)AIP-1  . In comp > 0.05) . Since ta5)AIP-1 .S. aureus MRSA ST239 strains identified agrC as among the best predictors of cytotoxicity . Introduction of the I311T substitution alone into the ROJ143 agrP3 reporter increased the EC50 from 2.8 ± 1 nM to 17 ± 3 nM (P < 0.01), while both I311T and A343T substitutions together increased it significantly further    .agr loci of two other ST239 strains lacking the I311T substitution (ICP5014 and ICP5062) but with markedly different cytotoxicities were also compared and found to be identical except for AgrC in ICP5062, which contained a T247I substitution. Although AIP-1 production was undetectable in ICP5062 until the stationary phase (53 nM after 7 h [P < 0.01)   . The int < 0.01) . Howeveragr function. 50s of 8 ± 2 nM (P < 0.05) for L245A and 8 ± 1 nM (P < 0.01) for F264A, respectively (S. aureus isolates are summarized in Table S5.Cytoplasmic domain AgrC substitutions in the same region as N267I which have previously been reported to affect exotoxin production include L245S  and F264ectively . The comThe structure of the AgrC cytoplasmic domain (residues 201 to 430) divided into DHp (dimerization and histidine phosphorylation) and CA  subdomains is illustrated in To gain further insights into their impact on domain structure, we generated homology models of the cytoplasmic domains of the AgrC wild type (WT) and F251Y, N267I, I311T, A343T, T247I substituted proteins, as well as the double mutants F251Y-N267I, T247I-I311T, I311T-A343T, and F251Y-N267I . These iS. aureus strains TS13 and TS14 and the WT AgrC CXR92122 is nearly isomorphic with the F251Y structure, although there is a subtle shift in the helices α1 and α2, as well as in the leading helix α3 of the CA subdomain. However, this shift did not affect the EC50 for AgrC activation  was further increased compared to the single AgrC I311T mutant (17.4 nM). This increase can be attributed to a significant alteration in the AgrA-binding domain where the structural change is most pronounced. Also, in this double mutant, the CA subdomain is brought forward toward the putative AgrA-binding domain and may interfere directly with binding.AgrC substitutions I311T and A343T reside within the CA subdomain . Althougc domain . The EC5The double mutation T247I-I311T is associated with a collapse of the helical hairpin onto the CA subdomain . This ocMutations T247I, F251Y, and N267I are located near the turn between helices α1 and α2 directly in the putative AgrA-binding region. Substitutions T247I and N267I kink the long helices α1 and α2, as well as altering their mutual orientation and interactions . In thisThe observed changes in domain organization within the cytoplasmic domain of AgrC can affect both global and local motional freedom in the polypeptide chain. Changes in local segmental mobility at individual amino acid residues that result from specific mutations, were followed via the root mean square deviation (RMSD) along the trajectory . Cumulative average excursions over the entire trajectory are summarized in Table S6. Overall, the cytoplasmic domain remains well structured, with little mobility over the majority of the helical hairpin and with cytoplasmic domain and RMSD values remaining within 2.3 to 4.6 Å. Notable flexibility is observed in the loop F386-L395 within the G box and the ATP-binding site, as well as the turn between helix α1 and helix α2 E250-L260.The local mobility of loop F386-L395 in A343T and I311T-A343T is reduced compared to the WT, whereas I311T alone significantly increases loop mobility. This shows that A343T alone is the deciding factor in restricting local loop dynamics that is sufficient to counter the increase in motional freedom resulting from substitution I311T . Specifically, flexibility within the WT loop is focused in GLG around L395, whereas in all other cases an increase in loop mobility is observed over a much longer stretch of the chain. Since this loop forms a major part of the ATP-binding pocket, changes in flexibility will likely affect both access of ATP to the binding site and ATPase kinetics.50 by 3- and 7-fold, respectively , determined between reporter residues K389 in the middle of the loop and E342 located on helix α4 , helix α4  and 7. Thelix α4 . The RDFhelix α4 . Also, aA bimodal distribution is also observed for mutation N267I in the helical hairpin region with a major closed loop at 6 Å and a parallel, also significant open population at 22 Å. A bimodal loop conformation is also apparent for T247I and F251Y-N267I with a very similar trajectory occupation, a closed conformation at 6Å and a more open conformation at 15Å. Interestingly, both F251Y and T247I-I311T appear closed at 6 Å. All cases of bimodal trajectory occupation observed in agrC genes that result in delayed AIP production and accumulation and reduced exotoxin but which nevertheless respond to exogenous AIP in a growth-phase-dependent manner. The agr system is subject to complex control by a number of regulators, repressors, and alternative sigma factors, including the Sar proteins, Rot, and MgrA  , the double mutant I311T-A343T resulted in a major repositioning of the dimerization hairpin with respect to the CA subdomain. Altered accessibility to the ATP-binding site, modulated by the flexibility and/or bistable conformational preference of loop E384-G394, could impact ATP binding as well as the efficiency of autophosphorylation, thus explaining the altered agr activity observed in the clinical isolates harboring the double mutation.Data from cytotoxicity, AIP, and mutant bioreporter assays collectively suggest that A343T and I311T have a cumulative effect on The conformational impact of point mutations in the cytoplasmic domain of AgrC appear delocalized, cooperative, and distributed over the entire domain, potentially affecting catalytic activity, dimerization, and downstream AgrA-mediated signaling. This points to a role for the cytoplasmic domain of AgrC as a virulence modulator rather than as a binary switch. Such fine-tuning in signal-mediated gene regulation provides evolutionary guidance and an opportunity for adaptation in response to environmental changes rather than serving as a definitive and committed survival response.This conformational analysis of point mutations in cytoplasmic AgrC, combined with studies of the whole protein , a chimeagr activity without completely abolishing activation. As a consequence, they could be seen as an ideal adaptation of clinical strains to host niches where high-level exotoxin production is not required but where some measure of flexibility would be useful. Interestingly, each of the AgrC substitutions investigated in the present study is dependent on a single base change such that reversion to the wild type would be readily selectable. It has also been hypothesized that agr mutants show reduced fitness for transmissibility or possibly the establishment of new infections  or CYGP broth (5)AIP-1 were synthesized as previously described  and sequenced with an Illumina MiSeq 2 × 150-bp paired-end run. Reads were mapped to the EMRSA-15 representative genome HO 5096 0412 (GenBank accession no. NC_017763.1) (https://www.ebi.ac.uk/ena).Genomic DNA was prepared from 17763.1)  with the17763.1) . SNPs we17763.1)  using deS. aureus ROJ143 is an agrP3::lux bioreporter strain constructed by replacing the entire agr locus in RN4220 with the erythromycin resistance gene ermB and an agrP3::luxABCDE promoter and transformed with plasmid pAgrP2C1A, containing the agrP2 promoter, agrC1, and agrA  before ligation of the resulting PCR products by Quick Ligase enzyme (New England Biolabs). The plasmids carrying the mutated agrC1 genes were introduced into ROJ48 , harboring the agr locus at an ectopic chromosomal site, were constructed by phage transduction. Plasmid pEJM6 was made by introducing the wild-type group 1 agrP2BDCA genes amplified with primers EJM63 and EJM65 (Table S2) into the integrative plasmid pLL102 (Table S1). pEJM6 was transformed into a chromosomal integration strain of S. aureus CYL12349  after normalizing to the same optical density at 600 nm. Precipitated exoproteins were separated on 12% (wt/vol) SDS-PAGE gels, transferred to a nitrocellulose membrane, and blocked in 5% (wt/vol) skimmed milk in phosphate-buffered saline (pH 7.4) prior to incubation with mouse monoclonal antibodies to PVL, followed by a protein A-horseradish peroxidase conjugate (Sigma) and developed using ECL substrate .Exoproteins from S. aureus TS13 and TS14 were grown in BHI broth until mid-exponential phase. RNA Protect (Qiagen) was added, the cells were lysed and RNA purified using an RNeasy minikit (Qiagen). cDNA was synthesized from RNA samples with Superscript II reverse transcriptase (Invitrogen) and amplified using a Power SYBR green master mix (Life Technologies Corp.) using the required forward and reverse primers (Table S2). Amplification was recorded using an AB7500 real-time PCR system (Applied Biosystems), and comparative cycle threshold values were calculated to determine the relative expression of the target genes lukS-PV and RNAIII, with gyrB as a constitutively expressed control.50 values were generated from sigmoidal dose-response curves.Lux-based bioreporters containing either wild-type or mutant agrP2C1A plasmids (Table S1) were grown overnight at 37°C in BHI with 10 μg/ml chloramphenicol, diluted 1:50 in fresh BHI, and grown for a further 2 h prior to dilution to 1:20 into a 96-well microtiter plate containing triplicate serial dilutions of exogenous AIPs. Bioluminescence was quantified in a Tecan microplate reader, and data were plotted as relative light units/optical density (RLU/OD) over time. Peak values from each concentration of AIP were exported to Prism 2 , and ECS. aureus cultures throughout growth in BHI, samples were removed at specific time points and incubated with the bioluminescent agr reporter strain ROJ143 (Table S1), and the relative AIP concentration was determined from a calibration curve generated using a range of synthetic AIP standards.To quantify relative AIP concentrations in S. aureus is identical to UniProt accession no. Q2VG18 and NCBI GenPept accession no. CXR92122. Structural information is unavailable for full-length AgrC membrane and cytoplasmic domains modeled separately, and only the C-terminal catalytic part of the cytoplasmic domain has been characterized (S. aureus (PDB 4BXI), tyrosine kinase from Caulobacter crescentus (PDB 4Q20), and C. vibrioides (PDB 5IDJ).The sequence of AgrC from group 1 cterized . We usedcterized  and Phyrcterized  to obtaiin silico mutagenesis and subsequent comparative analysis. Wild type was prepared for MD simulations and, in parallel, the annealed model was mutated in CHARMM-GUI.The models were in good agreement and were subsequently annealed by all atom molecular dynamics for 60 ns using NAMD  with theThe docked conformation of ATP in the cytoplasmic domain of AgrC was obtained from exploratory semirigid docking using Swiss-Dock , and theStaphylococcus aureus reference genomes used include HO 5096 0412 (GenBank accession no. NC_017763.1) and NCTC8325 (GenBank no. CP000253.1). Whole-genome sequencing data for this study are available in the European Nucleotide Archive (ENA no. PRJEB32550) for TS13 (ENA no. ERS3409631) and TS14 (ENA no. ERS3409632). AgrC protein sequences were accessed from UniProt (UniProt no. Q2VG18) and the NCBI Peptide Database (GenPept no. CXR92122). Crystal structures were obtained from the Protein Data Bank, including AgrC from S. aureus (PDB 4BXI), tyrosine kinase from Caulobacter crescentus (PDB 4Q20) and C. vibrioides (PDB 5IDJ).The


The manual classification of protein domains is approaching its 20th anniversary. ECOD is our mixed manual-automatic domain classification. Over time, the types of proteins which require manual curation has changed. Depositions with complex multidomain and multichain arrangements are commonplace. Transmembrane domains are regularly classified. Repeatedly, domains which are initially believed to be novel are found to have homologous links to existing classified domains. Here we present a brief summary of recent manual curation efforts in ECOD generally combined with specific case studies of transmembrane and multidomain proteins wherein manual curation was useful for discovering new homologous relationships. We present a new taxonomy for the classification of ABC transporter transmembrane domains. We examine alternate topologies of the leucine-specific (LS) domain of Leucine tRNA-synthetase. Finally, we elaborate on a distant homologous links between two helical dimerization domains. The classification of protein structures deposited in the PDB increasingly involves complexes, transmembrane proteins, and multidomain proteins with non-globular internal repeats . This trWe have previously described ECOD (Evolutionary Classification Of protein Domains), our comprehensive classification of protein structures and their domains . BrieflyHere we present five case studies of protein classification where automated approaches failed, but manual curation resolved the classification. These cases were chosen from a period of over 2 years of manual curation of ECOD and reflect the current state of the manual curation of our classification. Two cases illustrate the complications of the classification of transmembrane domains. The ABC transporter transmembrane domains are ubiquitous proteins with complex evolutionary origins. We present a case where a previous homologous link was judged insufficient for classification, as well as a novel structure which joined previously unlinked ECOD homologous groups. The final two cases reflect the difficulty of classifying homologous multi-domain proteins with variant topology within their conserved domain architecture. We show how we identified the Leucine-specific domain (LS) of leucine tRNA-synthetase (LeuRS) is topologically distinct between orthologs and likely a detiorated zinc ribbon domain descended from rubredoxin-like domains. Additionally, we judge that the dimerization domain of PAN3 is homologous to the human Caprin-1 dimerization domain based on topological and functional similarity.The mission of ECOD is the comprehensive classification of the proteins with known three-dimensional structures. Our target is the Protein Data Bank (PDB), the current global resource for the deposition of protein structures. Since the release of ECOD in 2014, we have pursued a policy of weekly updates, using a combined automated pipeline and manual curation workflow. Although our full automatic methodology is described elsewhere , 11, 12,We analyzed 2 years of weekly and semi-weekly updates between 2016 and 2018 to examine the outcome of our manual curation decisions. Over this period, representative proteins chains were selected from a set of proteins for which the automated domain partition and assignment pipeline failed to generate complete domain assignments. This representative set (70% sequence identity) conained 2298 protein chains contained within 1656 PDB depositions over 120 updates. The principal curation judgement is whether a protein chain contains non-domain regions. 1558 (67%) protein chains were wholly or in part were judged unclassifiable as domains. 435(18%) were entirely classified into an ECOD special architecture , whereas only 1123(48%) were classified in part as special architectures. Of these representative chains, 1351(58%) contained at least one region that was partitioned and assigned into a domain by manual curators. Additionally, 732(31%) protein chains classified by manual curators into domains consisted of only a single domain , whereas 619 (26%) protein chains were determined to be multi-domain proteins. 119 (5%) proteins requiring curation contained at least one transmembrane segment (as determined by PHOBIUS. 1335 (58%) were from structures determined by X-ray crystallography, whereas 802 (35%) were determined by electron microscopy. These decisions to split and assign representative chains are the primary source of the curation examples discussed below.Eukaryotic type II CAAX prenyl endopeptidases, also named Ras and a-factor converting enzymes (RCE1), are integral membrane proteins that catalyze the removal of the “AAX” tripeptide from the CAAX motif  after the prenyl attachment to the cysteine residue . DistantRemote homologs to CPBP family proteases were identified by sensitive profile-profile comparisons, including the eukaryotic γ-secretase subunit APH-1 and the bacterial PrsW proteases . These pABC (ATP-binding cassette) transporters are a diverse group of proteins or protein complexes that couple the energy generated from ATP hydrolysis to transport/translocate various cargo molecules . ABC traThree non-canonical ABC transporter TMD domains: FtsX, YjgP_YjgQ, and DUF1430 – have been classified in the Pfam clan BPD_transp_1 along with the type I importer TMD (Pfam family BPD_transp_1). The structures of members of the FtsX –24 and YThe six TM segments of YjgP_YjgQ, ABCG, and ABCA TMDs adopt the same topology as the core domains of APH-1, as suggested by structure similarity searches. For example, the DaliLite search using the YjgP_YjgQ TMD as a query  found the APH-1 structure  with a Z-score of 8.9, the best score other than other YjgP_YjgQ domains. However, these ABC transporter TMDs do not have the conserved polar residues observed in the active site of APH-1 and type II CAAX proteases. It is unclear that these ABC transporter TMDs and APH-1/type II CAAX are homologs. Given their functional dissimilarity, we chose not to classify them in the same X-group.The type I ABC importer TMD (Pfam: BPD_transpd_1) is structurally similar to MacB (Pfam:FtsX) if its first TM segment is considered to structurally occupy the position of the last TM segment of the core of MacB/YjgP_YjgQ/ABCG/ABCA (H: type II exporter) with a coupling helix Fig. , pink inThe multidrug and toxic compound extrusion (MatE) family includes integral membrane proteins that couple electrochemical gradients to export of metabolites across the cell membrane leading to multidrug resistance (MDR) in bacteria and animals and disease resistance in plants. MatE transporters belong to a larger multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) exporter superfamily . The MOPVibrio cholerae MDR efflux pump NorM (PDB: 3mku) adopts a duplication of six TM helices (TMs 1–6 and 7–12) arranged as two domains  adopts a similar core 12 TM topology as MatE  exemplified by the inward-facing ABCG5/ABCG8 heterodimer structure Fig. c. The reLeu for use in protein synthesis. LeuRS consists of a main enzymatic component  and four additional flexibly linked domains, one of which is the leucine-specific domain [E. coli LeuRS (PDB: 4AQ7). This domain forms a two-layer β-sandwich with a β-sheet  and a β-hairpin separated by a small α-helix between strands 2 and 3 . In this case, β-strands 1 and 5 are twisted around each other, the alpha-helix between strands 2 and 3 is significantly longer and additional alpha-helix is between strands 3 and 4. Despite these differences, structures of the two topologies can be superimposed  is a multi-domain class Ia aminoacyl-tRNA synthetase whose main function is to synthesize Leu-tRNAc domain . The leuc domain . Among kE. coli LeuRS [T. thermophilus LeuRS [Desulfotignum phosphitoxidans, NCBI ID: WP_006965092.1). The Rosetta model we chose as our representative showed that three cysteines and one histidine face inwards towards a putative zinc-binding site from the corresponding zinc-binding loops  , but thili LeuRS . Similarus LeuRS . We noteus LeuRS , we idenus LeuRS , we perfThe poly(A)-specific nuclease (PAN) complex catalyzes mRNA deadenylation, a process in RNA degradation wherein AMP is released from the 3′ poly(A) tail of the mRNA substrate . PAN conCaprin (cytoplasmic activation/proliferation-associated protein) is a small family of proteins that appear quite isolated in protein sequence space (ref? Why?). Caprin-1 participates in various cellular functions through its interactions with RNA and other proteins . RecentlAlthough the authors considered Caprin-1 DD to have no structural similarity to existing structures, we found that it exhibits significant structural similarity to the previously published PAN3 DD structures. For example, a Dali search using Caprin-1 DD as a query returns multiple good hits to ECOD domains of PAN3 DD . Indeed, PAN3 DD and Caprin-1 DD share a common topology composed of four major α-helices arranged in similar positions and angles Fig. . A singlThe classification of protein domains has shifted from a model where the identification of novel topologies and functions associated with those topologies is a principal goal, to a model where the most attention must be focused on large protein complexes, including those with transmembrane regions, and the boundaries and interactions of domains within these complexes. ECOD, our protein domain classification, shares an intellectual ancestry with other structural domain classifications. Our mixed manual/automatic classification pipeline, ensures that protein that cannot be classified automatically are curated manually. As part of this process, we here presented five cases initially identified by manual curators and their subsequent analysis to discover homologs. These cases were selected as representative of the types of difficult cases currently encountered by manual curators. We confirmed a previous prediction  regarding the homology between Rce-1 and APH-1. Based on novel structures, we identified novel homologs of the ABC transporter transmembrane domains, and reorganized their taxonomy in our classification. A novel case of topological variation among homologs was identified among the LS domains of LeuRS. Finally, we identified a case of distant homology between Caprin-1 and PAN3 dimerization domains. Each of these cases illustrates specifically the more general trend towards large complex structures and the problems of classification of these structures being altogether different from the problems of distinguishing between topologies of soluble globular single-domain proteins.https://gremlin2.bakerlab.org/struct.php?page=ccmb). CcmB chain A was queried against the PDB25 representative dataset using the DaliLite server  [Coordinates for the CcmB structure model dimer were downloaded from the GREMLIN structure website  . Top hitThe DaliLite web server was used to identify structurally similar proteins for type II CAAX protease homolog , APH-1 , and ABC transporters. For ABC transporters, we used only the transmembrane domains as the query, while other domains such as nucleotide binding domains and extracellular substrate binding domains were excluded. The PDBs, their chains and residue ranges are: FtsX , ABCG , ABCA , YjgP_YjgQ , BPD_transp_1 , and type I exporter .D.phosphitoxidans LeuRS we did using Rosetta software suite, version 3.9 [http://robetta.bakerlab.org). We generated 2000 predicted models which were clustered according to their structure similarity using Calibur [E.coli LeuRS leucine-specific domain from this cluster was selected to be shown at the Fig. For comparison of proteins, three-dimensional structures and their alignments were used DALI  and TM-asion 3.9 . For Ros Calibur . The lar


Bacteriodetes hosts. CrAssphage, the first crAss-like phage to be discovered, contains a protein encoding a Bacteroides-associated carbohydrate-binding often N-terminal (BACON) domain tandem repeat. Because protein domain tandem repeats are often hotspots of evolution, BACON domains may provide insight into the evolution of crAss-like phages. Here, we studied the biodiversity and evolution of BACON domains in bacteriophages by analysing over 2 million viral contigs. We found a high biodiversity of BACON in seven gut phage lineages, including five known crAss-like phage lineages and two novel gut phage lineages that are distantly related to crAss-like phages. In three BACON-containing phage lineages, we found that BACON domain tandem repeats were associated with phage tail proteins, suggestive of a possible role of these repeats in host binding. In contrast, individual BACON domains that did not occur in tandem were not found in the proximity of tail proteins. In two lineages, tail-associated BACON domain tandem repeats evolved largely through horizontal transfer of separate domains. In the third lineage that includes the prototypical crAssphage, the tandem repeats arose from several sequential domain duplications, resulting in a characteristic tandem array that is distinct from bacterial BACON domains. We conclude that phage tail-associated BACON domain tandem repeats have evolved in at least two independent cases in gut bacteriophages, including in the widespread gut phage crAssphage.The human gut contains an expanse of largely unstudied bacteriophages. Among the most common are crAss-like phages, which were predicted to infect  Bacteriophage populations are essential for stability and proper functioning of the human gut microbiome . Gut phaBacteroides-associated carbohydrate-binding often N-terminal (BACON) domains [Bacteroidetes bacteria, but despite their name, a direct role in carbohydrate-binding for BACON domains is yet to be proven. The only currently published BACON domain X-ray crystallography structure in human gut Bacteroidetes indicated that they form links between carbohydrate-binding domains and are anchored onto the surface of Bacteroides cells and do not perform a carbohydrate-binding function themselves [One characteristic crAssphage protein (gp64 in NC_024711.1) encodes a tandem repeat of eight  domains . BACON demselves . This X-emselves . This maemselves . Phage iemselves  and carbemselves , and areemselves ,17, p2 [emselves , HK620 [emselves , p22 [20emselves  and Sf6 emselves . LikewisProteins with domain tandem repeats are found throughout the tree of life . BecauseHere we studied the evolution of BACON domain tandem repeats in crAss-like phages. First, we constructed a profile hidden Markov model (HMM) of the crAssphage-like BACON domain (crAss-BACON), which we identified in half of the known candidate crAss-like phage lineages and in two novel gut phage lineages. Tandem repeats of the crAss-BACON were exclusive to two crAss-like phage candidate genera and one of the novel lineages, and were always genomically flanked by tail proteins. In phage lineages with single (non-repeated) crAss-BACONs, they were associated with the replicative and head regions of the phage genome. By studying crAss-BACON evolution, we showed that crAss-BACON tandem repeats are divergent from bacterial domains, whereas individually occurring crAss-BACON domains are more closely related to bacterial sequences, indicating frequent horizontal transfer between viral and cellular organisms. Finally, we showed that the crAss-BACON tandem repeat in the most widespread crAss-like phage lineage has evolved through multiple single-domain duplications.Bacteroidetes bacteria, was downloaded from the Pfam database v32.0 on 1 April 2019 [We identified BACON domains across a broad range of viruses using two datasets that included both genomic and metagenomic sequences. The first dataset consisted of 661 crAss-like phage sequences from five recent publications ,10,11,29ril 2019 . The 304All bioinformatics tools mentioned below were run with default settings and cut-offs, except where stated explicitly.Before searching for BACON domains, we predicted all ORFs in both datasets of viral sequences using Prodigal v2.6.1  with trap-values was constructed with the R function dist and used for clustering with the Ward.D2 algorithm as incorporated in R. The optimal number of clusters was determined using the NbClust v3.0 R package [To obtain a tentative taxonomical classification of the crAss-BACON-containing contigs, we clustered them based on shared protein content ,38,39. T package , whereas package .To analyse the conservation of the cluster 1 crAss-BACON tandem repeat, all cluster 1 crAss-BACON ORFs with eight domains were selected and aligned with Clustal Omega v1.2.1 . InformaMost crAss-BACON-containing contig clusters contained members of previously defined candidate crAss-like phage subfamilies and genera, except clusters A and B which consisted of highly dissimilar viral sequences (see Results and Discussion). To provide an initial sequence-based characterization of these novel viruses, the largest contigs from each cluster were extracted and annotated using the PROKKA v1.11 software tool , with thTo further study the relation of the newly described contig clusters to known crAss-like phages, we performed a phylogenetic analysis of crAss-like phage head proteins. Five crAss-like phage head proteins that were previously used to determine crAss-like phage taxonomy  were extHosts were predicted for all crAss-BACON contigs with a length over 75,000 bp using the host prediction algorithm WiSH v1.0 . This prE-value ≤10−5) to proteins with a “hypothetical protein” function description. Because this provided no additional insight into the role of these 121 crAss-BACON ORFs, they were discarded from the analysis. All other neighbourhoods were plotted using the ggplot2 v3.1.0 R package.To study the genomic neighbourhood of crAss-BACON ORFs, these plus five ORFs up- and downstream were collected. All ORFs were queried against the NCBI non-redundant protein sequences collection  with BLATo study crAss-BACON evolution, we constructed an approximate maximum likelihood tree. CrAss-BACONs were first aligned with Clustal Omega v1.2.1 . While cTo analyse whether the cluster 1 crAss-BACON array had evolved through simultaneous duplication of multiple domains, the eight domains from crAssphage were subjected to a BLASTp all versus all using BLAST+ v2.9.0 , and bitBacteroides-derived BACON domain that was available in Pfam (PF13004) [We started our study of BACON domain diversity and evolution by constructing a profile HMM distinctive to crAssphage-like BACON domains (crAss-BACONs). The PF13004)  containePF13004) ,27 out oPF13004) . These aAlpha-, Beta-, Gamma- and Deltacrassvirinae, but not in all ten candidate genera. Candidate genera I, II, III, V, VI and VII contained crAss-BACONs, whereas IV, VIII, IX and X did not. Alphacrassvirinae is the most widespread subfamily that contains candidate genus I and the prototypical crAssphage. This subfamily contains two candidate genera with crAss-BACONs (I and III) and two without it (IV and IX). BACON domains are particularly prevalent in candidate genus I, where 55 of the 63 genomes that were previously categorised in this genus contain crAss-BACONs. In candidate genus III, 7 of the 22 genomes contain crAss-BACONs. Likewise, Deltacrassvirinae contains one candidate genus with crAss-BACONs  and two without it (VIII and X). We found crAss-BACONs in Betacrassvirinae , which contains the first isolated crAss-like phage [Gammacrassvirinae . Thus, crAss-BACONs were absent from some lineages, even though the search space contained genome sequences from all established candidate crAss-like phage genera that a previous publication showed were (near-)complete [The dataset in which we searched for BACON domains included contigs from an earlier study that presented a preliminary crAss-like phage taxonomy . That stcomplete . We concn = 2), chicken ceca (n = 2), sheep rumen (n = 1) and wombat intestines (n = 1), as well as three contigs originating from human oral metagenomes [As BACON domains were found in taxonomically diverse crAss-like phages, we next determined how widespread they were in other phages. For this, we queried 2,147,193 viral sequences from seven metaviromics studies for the crAss-BACON profile HMM b and ideagenomes   and Klebsiella pneumoniae (14 ORFs). Multiple cluster B ORFs hit phage structural proteins located in bacterial genomes, including three in A. baumannii, one in K. pneumoniae, one in Desulfovibrio alaskensis and one in Rhodobacteriacea. Like the cluster A contig, the cluster B contig may thus have a temperate lifestyle. The high number of ORFs with hits in Proteobacteria, coupled with only three ORF hits to Bacteroidetes species, could indicate that this cluster B phage infects Proteobacteria, a different phylum than the crAss-like phages [Bacteroidetes as previously thought [Bacteroidetes hosts for cluster B contigs, consistent with crAss-like phages [To further analyse the phage lineages in clusters A and B, we functionally annotated the protein-encoding genes on the longest sequences from clusters A and B. These contigs were 154,123 bp (cluster A) and 87,116 bp (cluster B) long and contained 180 and 110 ORFs respectively . These phages infect four different phyla and are unrelated to crAss-like phages. The above results show that cluster A and B genomes likely represent newly described phage lineages.Few ORFs in either the cluster A or B contigs are homologous to phage proteins in the RefSeq database see . Of the After establishing that crAss-BACONs are widespread in crAss-like phages and beyond, we next looked at differences in crAss-BACON architecture among the phage clusters. Domain tandem repeats in cellular organisms tend to rapidly evolve following domain duplications, after which they remain stable and conserved within species . The tanOther crAss-BACON architectures were found outside of cluster 1 where all crAss-BACON-containing ORFs had fewer than eight domains. Most crAss-BACON ORFs in clusters 2, 5 and 6 contain a single domain a. MultipE-value of 6 × 10−8) and a putative T9SS sorting domain-containing protein , were solely based on an alignment to BACON domains. To circumvent this lack of detectable sequence similarity of crAss-BACON ORFs to proteins with predicted function, we instead analysed their genomic neighbourhoods , the genomic regions that contained crAss-BACON ORFs had high sequence similarity, whereas the rest of the phage genomes did not . This suTo investigate BACON domain evolution in phages and bacteria, we created a phylogeny of 1509 BACON domains a. This iThe crAss-BACONs in the cluster 7 and B tandem arrays showed a greater spread in the tree c,d. LikeAlphacrassvirinae candidate subfamily that contains the longest and most conserved crAss-BACON tandem array  belongsAlphacrassvirinae). In this lineage, we showed how a stable tandem repeat of eight crAss-BACONs has resulted from multiple single domain duplication events. The presented results show that focus on uncharacterised proteins can provide insight into the enormous biodiversity and evolutionary dynamics of the viral world.We investigated the evolution of BACON domain tandem repeats in crAssphage and other gut bacteriophages. We demonstrated that crAss-like phage BACON domains (crAss-BACONs) are widespread in the crAss-like phage family, and we identified crAss-BACONs in two novel gut phage lineages. We further found that crAss-BACON tandem repeats are associated with tail fibres in three gut phage lineages. In two lineages (clusters 7 and B), these repeats are the result of horizontal transfer and tandem duplication. The third lineage with tail-associated crAss-BACON tandem repeats includes the prototypical crAssphage (cluster 1, candidate subfamily


PYHIN gene family of cytokines, which are having signature N-terminal PYD  domain and C-terminal HIN-200  domain joined by a linker region. The positively charged HIN-200 domain senses and binds with negatively charged phosphate groups of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) purely through electrostatic attractions. On the other hand, PYD domain interacts homotypically with a PYD domain of other mediators to pass the signals to effector molecules downwards the pathways for inflammatory responses. There is remarkable inter-specific diversity in the numbers of functional PYHIN genes e.g. one in cow, five in human, thirteen in mice etc., while there is a unique loss of PYHIN genes in the bat genomes which was revealed by Ahn et al. (2016) by studying genomes of ten different bat species belonging to sub-orders yinpterochiroptera and yangochiroptera. The conflicts between host and pathogen interfaces are compared with “Red queen’s arms race” which is also described as binding seeking dynamics and binding avoidance dynamics. As a result of this never-ending rivalry, eukaryotes developed PRRs as antiviral mechanism while viruses developed counter mechanisms to evade host immune defense. The PYHIN receptors are directly engaged with pathogenic molecules, so these should have evolved under the influence of selection pressures. In the current study, we investigated the nature of selection pressure on different domain types of IFI16-like (IFI16-L) PYHIN genes in ruminants.ALRs (AIM2-like Receptors) are germline encoded PRRs that belong to IFI16-like gene were found in PBMCs of ruminant animals-water buffalo, zebu cattle, goat, and sheep. The IFI16-like gene has one N-terminal PYD domain and one C-terminal HIN-200 domain, separated by an inter-domain linker region. HIN domain and inter-domain region are positively selected while the PYD domain is under the influence of purifying selection.Three transcript variants of the IFI16-like PYHIN genes in the ruminants. This data can be useful to predict the molecular determinants of pathogen interactions.Herein, we conclude that the nature of selection pressure varies on different parts  of The online version of this article (10.1186/s12862-018-1334-7) contains supplementary material, which is available to authorized users. Toll-like Receptors (TLRs) and C-type Lectin Receptors (CLRs). PRRs which reside and function inside the cellular compartments include NOD-like Receptors (NLRs), RIG1-like receptors (RLRs) and AIM2-like receptors (ALRs)  of four ruminant species from National Dairy Research Institute (NDRI) animal herd and local slaughterhouse, as follows: IFI16-L genes were designed from the conserved regions of the predicted IFI16-L gene sequences of ruminants  from GenBank. Around 100 ng of cDNA was used as the template for each amplification reaction containing 0.5 μL Advantage Taq Polymerase , 10 pmol of each IFI16-L primers in a total of 25 μL reaction mix. The amplified PCR products were cloned using InsTAclone PCR cloning kit  and transformed into XL1-Blue competent E.coli cells (Stratagene). The recombinant clones were subsequently screened through PCR. Sanger’s sequencing was performed with DTCS quick start cycle sequencing kit (Beckman Coulter) and run on GenomeLab™ GeXP Genetic Analysis System (Beckman Coulter).Total RNA was extracted from 0.5 mL of collected blood samples (TRIzol reagent Method). The quality and quantity of RNA samples were determined using a NanoDrop 2000 spectrophotometer  and were further ascertained by agarose gel electrophoresis. One μg of each RNA sample was reverse transcribed using the RevertAid First Strand cDNA synthesis kit , by using random hexamer primers according to the manufacturer’s instructions. Oligonucleotide primers  at 37 °C for 1 h and afterward incubated with FITC-conjugated Anti-rabbit secondary antibodies under the same conditions. Dilution of both primary and secondary antibodies used was 1:50. The cells were further co-stained with 1 μg/mL of DAPI and examined under fluorescent microscope.Buffalo fetal fibroblasts were cultured for 24 h at 37 °C with 5% CO2 and the next day the cells were fixed with 4% paraformaldehyde for 30 min at 4 °C. Cells were incubated with the Anti-Homologs of IFI16-L gene were searched with NCBI BLASTn tool and UCSC-BLAT. Additional mammalian PYHIN nucleotide sequences were retrieved from NCBI Database for phylogenetic analysis , which is taken as an outgroup; while in the case of inter-domain phylogenetics tree, Orcinus orca (cetacean), is taken as an outgroup. All the nodes having bootstrap values of < 50% were collapsed. Trees were exported in the Newick format and visualized using iTOL online software [Nucleotide sequences of PYD domain, HIN-200 domain and inter-domain  were selected. The PYD domain, HIN domain and the inter-domain linker region nucleotide sequences were aligned separately with the RevTrans 2.0 utility and the codon sequences were subsequently used for selection pressure analysis. Amino acid sequence alignments of PYD domain, HIN domain and inter-domain region are provided in Additional file To determine the selective force on Prior to selective pressure analysis, we checked all of the groups of nucleotide sequences for intragenic recombination. Recombination in sequences was tested with a difference of the sum of squares (DSS) method using a step size of 10 bp and a sliding window of 80 bp  as implep-values/posterior probabilities were taken into account for each analysis. Maximum likelihood tests were performed utilizing the partitioning approach for robust inference of selection (PARRIS) method [p-value < 0.05 (MEME), p-value < 0.1 (FEL) and p-value < 0.1 (PARRIS).We used statistical tools from the HyPhy package  implemen) method , which i) method , 48 were) method  was usedAdditional file 1:PYHIN genes. Orthology in PYHIN genes is not conserved. The number and type of PYHIN genes varies in different mammalian orders. Here, the PYHIN genes and their associated domains from selected orders of classes Mammalia, Reptilia and Sarcopterygii (fish) have been presented. The residue lengths of PYD and HIN domains of all the animals are conserved. Drastic variation in the length of the inter-domain linker regions (shown in grey colour) can be observed among the various animals as well as among the different PYHIN genes within the same animal. The small HIN region within the IFI16-L from the elephant represents a rudimentary form of HINa domain. (JPG 345 kb)Diversity of Additional file 2:Tables containing PARRIS results of (a) PYD domain, (b) inter-domain and (c) HIN domain. (DOCX 17 kb)Additional file 3:PYHIN sequences used for phylogenetic analysis. (DOCX 13 kb)Additional file 4:Multiple sequence alignments of (a) PYD domain, (b) inter-domain and (c) HIN domain. (DOCX 101 kb)


Salmonella enterica, these binding boxes are conserved within phylogenetically-related PAPs, such as AcrA and AcrE, while differing considerably between divergent PAPs such as MdsA and MdtA, despite overall conservation of the PAP structure. By analysing these binding sequences we created a predictive model of PAP-RND interaction, which suggested the determinants that may allow promiscuity between certain PAPs, but discrimination of others. We corroborated these predictions using direct phenotypic data, confirming that only AcrA and AcrE, but not MdtA or MsdA, can function with the major RND pump AcrB. Furthermore, we provide functional validation of the involvement of the binding boxes by disruptive site-directed mutagenesis. These results directly link sequence conservation within identified PAP binding sites with functional data providing mechanistic explanation for assembly of clinically relevant RND-pumps and explain how Salmonella and other pathogens maintain a degree of redundancy in efflux mediated resistance. Overall, our study provides a novel understanding of the molecular determinants driving the RND-PAP recognition by bridging the available structural information with experimental functional validation thus providing the scientific community with a predictive model of pump-contacts that could be exploited in the future for the development of targeted therapeutics and efflux pump inhibitors.Active efflux due to tripartite RND efflux pumps is an important mechanism of clinically relevant antibiotic resistance in Gram-negative bacteria. These pumps are also essential for Gram-negative pathogens to cause infection and form biofilms. They consist of an inner membrane RND transporter; a periplasmic adaptor protein (PAP), and an outer membrane channel. The role of PAPs in assembly, and the identities of specific residues involved in PAP-RND binding, remain poorly understood. Using recent high-resolution structures, four 3D sites involved in PAP-RND binding within each PAP protomer were defined that correspond to nine discrete linear binding sequences or “binding boxes” within the PAP sequence. In the important human pathogen  Salmonella and E. coli based on structural analysis and functional data. The analysis of conservation within these residue ranges allowed prediction of which PAPs that are able to function with the major efflux pump, AcrB. Further direct validation of the critical roles of these residue ranges by site-directed mutagenesis, allowed us to dramatically narrow the PAP-binding surface determinants. Overall, our study provides a novel understanding of the molecular determinants driving the RND-PAP recognition by bridging the available structural information with experimental functional validation thus providing the scientific community with a predictive model of pump-contacts that could be exploited in the future for the development of targeted therapeutics.Active efflux due to tripartite efflux pumps is an important mechanism of clinically relevant antibiotic resistance in Gram-negative bacteria. In this study, we have defined for the first time the residue ranges of the PAPs that are involved in the assembly of the major tripartite efflux systems in  Salmonella, Escherichia coli and other Enterobacteriaceae  tha tha27] tSalmonella. The PAPs have a hairpin-like arrangement with the polypeptide chain beginning at the inner membrane, crossing the periplasm to bridge the outer membrane component of the efflux complex in the middle of the protein and then folding back on itself with the C-terminal being located close to the N-terminal at the inner membrane  of the main RND protomer  or β-barrel of PAP 2 to the DC domain of the neighbouring RND protomer .Site 2: PAP β-barrel domain to β-hairpin of the RND funnel domain .Site 3: PAP MP-domain to base of RND funnel domain.Site 4: PAP MP-domain to RND pore (porter)-domain .We analysed the available cryo-EM data, of assembled AcrA-AcrB complexes, to define possible interacting residues. PAPs bind RND transporters in a 2:1 stoichiometry. This results in two protomers of the PAP binding to one protomer of the transporter at different semi-equivalent binding sites with slightly different specificities and affinities . Here weIt is striking, that despite the extensive surface area available for a tight interaction between the PAP and the transporter, the two proteins seem to have relatively limited contact, with the main stabilising interactions being restricted to the self-association of the lipoyl domains of the PAPs. This observation by itself directly leads to the suggestion, that from the relatively few remaining contacts, a number will have to be generally preserved for structural rigidity and may be conserved in nature across the PAP-transporter pairs, while a smaller number still will play the role of “discriminators” requiring exact pairing between the PAP and its cognate transporter.E. coli AcrA on Taking these considerations into account and to simplify the analysis of the binding sites we have divided them into their actual linear sequence constituents and numbered them from N-terminus to the C-terminus of the PAP (using AcrA as a template). This resulted in 9 discrete “binding boxes” to which we will refer further in the text. They are visualized in the structural alignment  and mappSalmonella. We have previously reported that the PAPs in S. enterica display some promiscuity [Salmonella is an excellent model that enables study not only of drug resistance phenotypes, but also the effect of efflux on infection. At present there is no direct structural data available for any of the five RND pumps or the four PAPs in the important human pathogen Salmonella. Even for E. coli, structural information for RND-pumps other than AcrAB is not available. Therefore, homology models of Salmonella PAPs were designed to allow for structural analysis and sequence conservation mapping. We were able to construct reliable models of Salmonella AcrA based on the direct correspondence of the sequence between it and the experimentally determined partial E. coli structures [P. aeruginosa [To understand more about how these PAP “binding boxes” are conserved amongst the PAP family we conducted further work in miscuity  and Salmructures , as wellructures  and the ruginosa .Salmonella PAPs have the typical four-domain organization of RND-associated PAPs  did not alter antimicrobial susceptibility nor effect the rate of efflux of ethidium bromide  had the same antimicrobial susceptibility phenotype as the wildtype strain  did not further change the phenotype compared to the double acrA acrE mutant in terms of antimicrobial susceptibility or efflux rate  although the mutant selection frequency was not significantly different from that of the single acrA knockout   . Single ed to WT . Importal 4 PAPs .RND efflux is also required for biofilm formation so the ability of the mutants to form biofilm was added. None of the PAP mutants tested had a significantly altered ability to form biofilm in our model .acrA and acrE was additive but this was not true for mdtA or mdsA.Together these data support the structural analysis suggesting interoperability of AcrA and AcrE but not MdtA and MdsA because the effect of inactivating Δ4PAP strain was separately complemented with plasmids encoding one of the four PAPs. Under standard laboratory conditions the major pump AcrB is expressed at much higher levels than any of the other RND pumps and inactivation of the other pumps does not alter antimicrobial susceptibility, so any complementary effect seen following PAP expression seen will be meditated by forming a complex the AcrB pump.To investigate the hypothesis that AcrA and AcrE would be interoperable, but that MdtA and MdsA would not, the Δ4PAP strain with pET20b acrA increased MICs of most antimicrobials compared with the Δ4PAP strain although not to wildtype levels  was cloned into a higher copy plasmid (pTRC) and this revealed different patterns. As previously described, very high level over-expression of acrA was tolerated poorly by the cell, causing slow growth rate, filamentation and no phenotypic complementation  and M15 were sequenced and revealed a 36bp duplication including part of the DNA binding region ramR in M15  did not alter the susceptibility to the antimicrobials tested compared to the M15 parent suggesting that the high level of AcrE protein present must be working with the AcrB pump to provide efflux function  and non-conserved residues (expected to act as discriminators between different PAP-transporter pairs) by site directed mutagenesis followed by quantitative EtBr efflux assay  and measStructural mapping of the mutations revealed that with the exception of the G363 (box 9), the rest of the detrimental mutations belong to β-barrel domain residues of the PAP forming a tight cluster around the β-hairpins (DN and DC respectively) of the AcrB funnel domain, which provides the largest buried surface on the PAP-RND complex. Consistent with this, the mutations within rest of the boxes, that are less conserved and make lower number of interactions with RND protomers had very limited impact on the efflux function.The RND efflux pumps are an attractive target for inhibition due to their crucial roles in antibiotic resistance, virulence and biofilm formation e.g. , 9, 47. Salmonella and that this may have implications for future efflux inhibition strategies [binding boxes” which are readily identifiable in multiple sequence alignments  docking mode to MexB based on the structure of the complex which became available after the initial submission of this work [Pseudomonas and Salmonella, the structural alignment shows that the boxes align perfectly between the genera and furthermore critically important conserved residues have retained their positions within them.While these observations have strongly supported the proposed role of the binding boxes, we furthermore checked the predictive power of our model, by performing an additional “blind” analysis of the MexA from his work . As showbinding boxes, provides a testable hypothesis and a useful framework for further study of PAP-transporter interaction, and while the extensive mutagenesis needed to validate all of them as functional interaction sites goes beyond the remit of the current study, by defining them we were able to provide startling rationalisation of already available data, which lends strong support to this interpretation. For example, previous reports suggest that while Pseudomonas PAP MexA isn’t able to interact with TolC, thus rendering the chimeric MexAB-TolC pump inactive [E. coli AcrA appears to be rather promiscuous and capable of partial interaction with the Pseudomonas RND transporter MexB, and that interaction can be further improved by point mutagenesis [binding box 3”. The recent cryo-EM structures reveal that the AcrA residues 249–250 are in sufficient vicinity of the RND-transporter to engage in direct contact. Specifically, in 5O66.pdb D732 and K735 and the carbonyl of A803 provide plausible interaction partners from the side of the AcrB to PAP2. This is further reinforced by the observation that one of the MexB adaptive mutations, namely A802V, is located in a position equivalent to the one of A803 in AcrB ; and binding box4  which are revealed by current assembly structures to be in contact with the β-hairpins 1 and 2 of the AcrB DN and DC domains respectively, thus confirming that these binding boxes are directly involved in the pump recognition and assembly [Furthermore, previous data on genetic assessment of the role of β-hairpins in the DN and DC domains of AcrB identified compensatory mutations in AcrA, located within binding box 2  had no further effect than loss of just AcrA and AcrE and only expression of AcrA or AcrE was able to rescue the mutant phenotype of a Δ4PAP strain while MdtA and MdsA were not. Together this supports the hypothesis that conservation/discrimination based on the predicted binding residues translates into promiscuity or interoperability and redundancy of PAPs/pumps and directly affects the drug susceptibility profile.The described promiscuity of AcrA and AcrE may explain why we and others have found subtly different phenotypes from inactivation of AcrA and AcrB or both AcrAB e.g. . This woSalmonella and selection for increased expression of homologous efflux systems has also been described in E. coli [In addition these data further support the idea that the PAPs could be an effective target against which to develop efflux inhibitors because inactivation of them increased susceptibility to a range of antimicrobials; reduced the frequency at which mutants with other resistance mechanisms could be selected, and reduced virulence. However, we also found that AcrE over-expression could easily be selected for and phenotypically compensate for the loss of AcrA in  E. coli , 57. Thi“binding boxes”. We further validated these by showing that PAPs with conserved binding boxes were interoperable while those with a more divergent sequence were not. The discrete nature of the binding sites provides a promising rationale for targeting them with inhibitor molecules and thus decoupling the pumps. This information could also be exploited for creating “designer pumps” with defined engineered characteristics e.g. for improved bioethanol production. Combined with functional in vitro analyses, our results suggest a role for PAPs’ β-barrel and MP domains in vetting productive multidrug-efflux complexes. In addition our analysis highlights regions of PAPs critical for the transport mechanism of RND pumps in general; rationalizes previous gain-of-function mutations, and provides the structural basis of PAP-RND recognition, understanding of which will be important for future inhibitor design.To summarise, in this study we have mapped the residues required for binding of the PAP to the RND pump, identifying critical residues forming discrete https://mafft.cbrc.jp/).Multiple sequence alignments (MSA) were prepared using MAFFT and NJ/UPGMA phylogeny algorithms as implemented in MAFFT v.7 server (http://curie.utmb.edu/) using the available cryo-EM structures  and the results were further cross-validated manually using Coot [Structural annotations of the MSA sequences were done with Espript 3 . The Acring Coot . Sequencing Coot . Additioing Coot  in manuaSalmonella AcrA was modelled based on the direct correspondence of the sequence between it and the experimentally determined partial E. coli structures  [P. aeruginosa  [E. coli structures: 5O66:G.pdb and 5NIL:G.pdb. Due to the lower level of sequence identity, MdsA was modeled using the full-length MexA (2V4D:C.pdb). 53–298)  as well  53–298)  and the  chainC) . The temacrA, acrB and acrE mutants were constructed previously from Salmonella enterica serovar Typhimurium strain SL1344 [acrA or acrE, were constructed previously and vectors overexpressing mdtA and mdsA were generated according to manufacturer's instructions (Invitrogen pTrcHis). Strains were grown in Luria–Bertani (LB) broth at 37°C with shaking unless otherwise stated.The n SL1344 , 47, 63.n SL1344 . The PAPThe agar doubling dilution method was used to determine the MICs of various antibiotics and dyes according to CLSI guidance. All MICs were repeated at least three times and where necessary a modal value is used. All compounds tested were obtained from Sigma, UK.The efflux activity of the mutants was assessed by determining the accumulation of the fluorescent dye Hoechst H33342  as described previously .Efflux activity was also assessed by incubating cells in the presence of ethidium bromide and CCCP. Cells were re-energised and the rate of reduction in fluorescence was measured as previously described .600 nm of 0.6, cells were re-suspended in 50 mM potassium phosphate buffer and a viable count was taken. Fluorescence was read using a black microtitre tray in a FLUOstar Optima at excitation and emission wavelengths of 280 and 440 nm, respectively. The fluorescence reading was compared to a standard curve and then divided by the viable count to give amount of ciprofloxacin per cell. Data presented are the mean of three independent biological replicates ±SEM.Uptake of ciprofloxacin was measured as previously described  with theG. mellonella) larvae were purchased from Livefood UK Ltd.  and were maintained on wood chips in the dark at 14°C. They were stored for not longer than 2 weeks. Bacterial infection of G. mellonella was performed essentially as described by Wand et al [G. mellonella were injected with a bacterial load of approximately 1 x 104 CFU. The data were analyzed by the Mantel-Cox method using Prism software version 6 .Wax moth  were injected intraperitoneally (i.p.) with 3x103 CFU. The exact injected dose was confirmed by plating dilutions of the cell suspension used for infection on LB agar plates. The mice were sacrificed at 3 days post infection and spleens and livers were retained for analyses. To determine the bacterial burden in mouse organs, weighed liver and spleen sections were passed through a 70-μm nylon cell strainer  with 1 to 5 ml of PBS. The collected cell suspensions were diluted in PBS and plated onto LB agar plates without selection. The recovered colonies were counted and the bacterial burden per whole organ was calculated.Wild-type BALB/c mice were purchased from HO Harlan Olac Ltd . The mice were maintained under standard animal housing conditions in accordance with local and UK Home Office regulations. Overnight cultures of Salmonella strains for infection studies were inoculated into fresh LB medium at a 1/20 dilution and grown at 37°C to an ODThe ability of mutants to form biofilm was measured using the crystal violet method as described in .acrA, ΔacrAE and ΔacrAΔacrEΔmdsAΔmdtA (Δ4PAP) as previously described [Mutant selection experiments were performed with strains SL1344, Δhttps://github.com/tseemann/snippy).DNA extraction and WGS was performed by microbes NG, Birmingham. SNPs were identified relative to SL1344 using Snippy v3.2 (RNA isolation and qRT-PCR were performed as previously described  except tSite directed mutagenesis was performed using QuikChange XL mutagenesis kit (Qiagen) and the StAcrA-pET20b template was used to introduce the mutations. The mutant and WT AcrA proteins were expressed without induction using the leaky background expression in the Δ4PAP derivative of the SL1344 strain as described above.600 of 0.7. Cells were harvested and lysed in 50mM Tris-HCl 7.5, 200mM NaCl and 10mM β-DDM (Anatrace), supplemented by Completed EDTA-Free proteinases inhibitor tablets (Roche) using Emulsiflex cell disruptor. Cell debris were removed by centrifugation at 20,000g and the supernatant separated using 4–12% Bis-Tris precast gradient SDS-PAGE gel (Invitrogen). Following run the gel was transferred onto PVDF membrane and visualised by anti-6xHis-tag AP-conjugated antibody (Abcam) and visualised using NBT/BCIP chromogenic substrate (Sigma).For the purposes of expression testing we introduced a C-terminal His-tag (by QuikChange as above) into the StAcrA-pET20b harbouring the mutated versions of the AcrA gene. The constructs were transformed into Δ4PAP derivative of the SL1344 and protein production induced with 0.5mM IPTG at ODS1 FigRND transporters, of which AcrB is a prototypical member, are homo-trimeric proteins , 69. In (TIF)Click here for additional data file.S2 FigA. Side-by-side comparison of the modelled Salmonella PAPs. Due to the lack of reliable structural templates, the N-terminal and C-terminal extensions of MdsA and MdtA  have not been modelled. B. Superposed models of the core 4 domains of AcrA (red), AcrE (blue) and MdtA (blue) show the closely matched fold and a predicted RMSD of below 1.3 Å for the entire C-alpha trace.(TIF)Click here for additional data file.S3 FigA. Superposition of PAP 1 and PAP2 protomers (on the example of AcrA 5o66.pdb chain G and chain H—in blue and red respectively), demonstrating the discrepancies of relative β-barrel domain and MDP domain orientations. Over the whole chain the RMSD is ~1.3 Å; over the MP domain 0.91 Å; β-barrel domain shows the highest individual discrepancy ~1.16 Å; while α-hairpin and lipoyl domains display 0.55 and 0.66 Å RMSD respectively. B. PAP 1 and PAP2 orientation superposed over β-barrel domain and MDP respectively.(TIF)Click here for additional data file.S4 Figbinding box” interfaces relative to the transporter is evident.Superposition done over the C-alphas of the β-barrel and MDP domains respectively. The preservation of the predicted “(TIF)Click here for additional data file.S5 FigA. survival of the Galleria wax moth larvae, the ability to B. infect the mouse model of infection and C. form biofilm. In the mouse model experiments six animals were infected for each mutant over two independent experiments and the data pooled. Data were analysed using the Mann Whitney test. The biofilm data is shown as the mean of three independent biological replicates +/- standard error.The effect of PAP inactivation on (TIF)Click here for additional data file.S6 FigA. Efflux of ethidium bromide. Bacteria were treated with ethidium bromide and CCCP for 60 min and then re-energized with glucose. Data presented is the time taken for the fluorescence to decrease by 25% +/- SE. When produced on pTrc acrA, mdsA and mdtA do not complement the mutant phenotype. B. Gram stains showing filamentation of the Δ4PAP strain when acrA is overproduced at a high level but not when acrE is produced at a similar level. C. Intracellular accumulation of ciprofloxacin. Data is displayed as the mean of at least three biological replicates each in technical duplicate + SE mean. * denotes a strain with a significantly different accumulation of ciprofloxacin compared to SL1344 (p<0.05 following a two way ANOVA with Dunnett’s multiple comparison test).(TIF)Click here for additional data file.S7 FigSalmonella AcrA and their effect are represented by star signs and colours. The alignment demonstrates that despite the divergent nature of the MexA and the presence of deletions in the sequence (e.g. at positions 117 and 135 corresponding to the hairpin domain) the overall positions and sizes of the boxes are identical, and furthermore, the positions of residues with pronounced effect on efflux are coinciding with the highly conserved residues within the boxes.The secondary structural elements and the corresponding sequence numbering are based on the structure 2V4D.pdb (UniProtKB P52477). The relative positions of the residues belonging to the binding boxes mutated in the (TIF)Click here for additional data file.S8 Fig(TIF)Click here for additional data file.S9 Fig(TIF)Click here for additional data file.S10 FigA: comparison of the PAP1 and PAP2 assembling on the CusA surface (left) and AcrB surface (right). Note the clear difference in PAP2 interaction with PC1 and PN2 domains. The lipoyl domains in CusBA complex also are much more vertically extended and present a steeper angle relative to the funnel domain of the transporter. B: Superposition of the PAP2 orientation in AcrAB complex (red) and CusBA complex–yellow. For clarity the beta-hairpin domain is removed. While beta-barrel domains are in similar orientation the linker to the MP domain seems to have undergone a large conformational change as a result of which the MP domain is interacting primarily with PC1 domain, but appears to have lost the PN2-domain interactions. Notably the N- and C-termini of the PAP2 in CusBA complex form extended contacts with the cleft between PC1/PC2 subdomains, which is homologous to the entry 2 tunnel of the drug efflux transporters.(TIF)Click here for additional data file.S1 TableData presented is the mean of at least three independent biological replicates +/- SEM.(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.