Mounting evidence suggests that the FAK N-terminal (FERM) domain controls FAK phosphorylation and function; however, little is known regarding the role of the C terminal (FAT) domain in FAK regulation. We identified a patient-derived FAK mutant, in which a 27-amino acid segment was deleted from the C-terminal FAT domain (named FAK-Del33). When FAK-Del33 was overexpressed in specific tumor cell lines, Y397 phosphorylation increased compared with that observed in cells expressing FAK-WT. Here, we attempt to unveil the mechanism of this increased phosphorylation. Using cell biology experiments, we show that FAK-Del33 is incapable of co-localizing with paxillin, and has constitutively high Y397 phosphorylation. With a kinase-dead mutation, it showed phosphorylation of FAK-Del33 has enhanced through auto-phosphorylation. It was also demonstrated that phosphorylation of FAK-Del33 is not Src dependent or enhanced intermolecular interactions, and that the hyperphosphorylation can be lowered using increasing amounts of transfected FERM domain. This result suggests that Del33 mutation disrupting of FAT's structural integrity and paxillin binding capacity leads to incapable of targeting Focal adhesions, but has gained the capacity for auto-phosphorylation in cis. Focal adhesion kinase (FAK) plays a key role in promoting adhesion, proliferation, and cell migration and in regulating anchorage-dependent anti-apoptotic signals Auto-phosphorylation at Y397 is a key event in FAK activation Selective FAK activation depends on the structure of FAK. FAK contains an N-terminal FERM domain, a central tyrosine kinase domain (KD), a C-terminal focal adhesion targeting (FAT) domain, a linker region between the FERM domain and the KD, and an unstructured proline-rich (PR) region between the KD and the FAT domain in vitro and in vivo. These researchers suggest that the structural dynamics of the FAT domain may play an important role in determining FAK function.The C-terminal FAT domain has multiple protein-protein interactions sites (residues 919-1039 in human and murine FAK) that are critical for the recruitment of FAK to focal adhesion complexes, and Tyr925 is located within this domain In a previous study, we identified the novel splicing mutation FAK-Del33 in both breast and thyroid cancers through colony sequencing. The FAK-Del33 mutation frequency is 14.2% (3/21) in breast carcinomas Homo sapiens, which suggest that these transcripts may play a regulatory role in transcription. Unlike these alternative variants, FAK-Del33 displays a relatively high abundance in tumor tissue, with 30-50% of samples containing mutant transcripts. These results indicate that FAK-Del33 may be related to cancer progression.Several alternative exons coding for short peptides have been reported in rat and mouse FAK FAK tyrosine phosphorylation plays an important role in regulating FAK-mediated signaling. In the present study, we report that FAK phosphorylation at Y397 was increased in several tumor cells. FAK activation can be achieved through Src-dependent integrin or growth factor receptor stimulation or through trans-phosphorylation by other kinase MDA-MB-435s, MDA-MB-468, MDA-MB-231, HepG2, Hep3B, NIH3T3, Cos7, and REF cell lines were purchased from ATCC  and cultured as recommended.For the mutation analysis, fresh tissues  from breast cancer patients were collected after surgery and subjected to RNA extraction using the Trizol reagent . RT-PCR was used to amplify the coding region of oligo-dT primed FAK cDNAs from the tissue specimens, and the products were cloned into pCR2.1 vectors. Ten positive colonies were selected from each sample and sequenced.To illustration of the 3D structure of the FAT domain, the software of pymol was used. For the FAK-WT and FAK-Del33 amino acid residue alignments, the online software of BLAST (bl2seq) was used. The reference FAK cDNA sequence was downloaded from CCDS Sequence Data for PTK2 [Homo sapiens (human)] with Gene ID: 5747 in NCBI website.The pCR2.1-FAK-Del33 plasmid, which contains the human FAK splicing mutation sequence fused to an HA tag, has been previously described Cells were co-transfected with pSV2neo, pTet-FAK, or pTet-FAK-Del33, or with the empty pTet-Splice vector as a negative control. G418-resistant colonies were cloned and tested for inducible FAK expression. The cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 300 mg/ml penicillin, 100 mg/ml streptomycin, 10% calf serum, and 5 ng/ml Dox (to suppress FAK expression). FAK expression was induced by growth without Dox for 16–18 h prior to the experiments.+-GFP plasmid was a generous gift from Dr. Peihua Ni (University of Shanghai Jiaotong). DNA containing the entire FAK-Del33 coding sequence flanked by KpnI and NotI sites was generated from the pCR2.1-FAK-Del33 plasmid by PCR using Taq polymerase and the cloning primers are listed in +-GFP vector to generate GFP-FAK-Del33 plasmids, which expresses GFP fused to the C-terminal of FAK-Del33.The PCDNA3.1+, and this plasmid resulted in overexpression when transiently transfected.Using the same strategy, pCR2.1-FAK-Del33 was cleaved with NotI and NheI and then sub-cloned into PCDNA3.15′-ATA AGC GGC CGC TCA AAT CTC AGC ATA ATC ATC TGT-3′). The other PCR fragment was generated by amplifying pCR2.1-FAK-WT/pCR2.1-FAK-Del33 with FAK 5′ BamHI  and the corresponding reverse mutagenesis primer (FAK Primer 2 5′-AGC AAA ATC ATC TGT TTC TGA CAC AGA GA-3′). The two purified fragments were mixed and amplified using the FAK 5′ BamHI and FAK 3′ NotI primers, and the resulting fragment was cloned into pCDNA3.1+.FAK-Del33 containing the Y397F mutation was generated by two-step PCR as follows . One PCR fragment was generated by amplifying pCR2.1-FAK-Del33 with the forward mutagenesis primer FAK Primer 1  and FAK NotI (5′-ACA TGT CCT AAT TGC AAC CGC CAA AGC-3′). The FAK/Y397F or FAK-Del33/Y397F plasmid was used as a template for the K454R mutation. The resulting fragment was cloned into PCDNA3.1+.A similar mutagenesis strategy was used for K454R . The forward mutagenesis primer was FAK Primer 3 (5′-CGG TTG CAA TTA GGA CAT GTA AAA ACT GT-3′), and the corresponding reverse mutagenesis primer was FAK Primer 4  and FAK 3′ NotI. The resulting PCR fragment was cloned into pCDNA3.1+.The expression of FAK cDNA for transient transfection was created by amplifying pCR2.1-FAK-WT or pCR2.1-FAK-Del33 using the following primers: FAK 5′ BamHI (5′-GTC GGA TCC ATG TTG GCC AAC AGC GAA AAG CAA GGC ATG-3′) and FAK NotI primers. The resulting PCR fragment was cloned into pCDNA3.1+.The FAK deletion mutants Δ375-FAK and Δ375-FAK-Del33 were created by amplifying pCR2.1-FAK-WT or pCR2.1-FAK-Del33, respectively, using the Δ375F  and the FAK NotI primer. The resulting PCR fragment was cloned into pCDNA3.1+.The 1–396 FAK (FERM) fragment was created by amplifying pCR2.1-FAK-Del33 using the FAK 5′ BamHI primer . Anti-HA and anti-FLAG epitope (DYKDDDDK) monoclonal antibodies were purchased from Sigma-Aldrich.Proteins were extracted from cells using lysis buffer for Westernblotting  supplemented with 1% protease inhibitor cocktail , 25 mM NaF, and 1 mM NaFor experiments where Cells replated on FN, ColI or poly-L-lysine were added to plates coated with 10 µg/ml FN, or 2 µg/ml Col I or 100 µg/ml poly-L-lysine for 60 min after being held in suspension. For experiments where cells were treated either the Src inhibitor PP2 or the control compound PP3, cells were plated onto FN-coated plates and transfected with indicated plasmids for 24 h. After that, Src inhibitor PP2 or the control compound PP3 were added to these cells for further 12 h before being lysed.Cells were transfected with 1 µg of DNA per well in 12-well plates, or with 2 µg of DNA per well in 6-well plates using Lipofectamine 2000  according to the manufacturer's instructions. Twenty-four hours after transfection, the cells were plated on glass coverslips and then fixed 24 h later for 20 min with PBS containing 4% (w/v) paraformaldehyde. After three rinses in PBS, the cells were permeabilized for 10 min with 0.2% Triton X-100 in PBS, treated with blocking buffer (PBS containing 5% w/v BSA) for 30 min and then incubated overnight with antibodies against paxillin or p-paxillin (Y118) at 4°C in PBS containing 1% BSA. After three rinses, the cells were sequentially incubated for 1 h at room temperature with goat Alexa-555-coupled anti-mouse antibodies. After three rinses, the cells were stained with DAPI . Images were acquired at the Cell Imaging Facility using a laser scanning confocal microscope (Leica and SP5).P-value was less than 0.05.The statistical significance between the data sets was determined using Student's t-test (SPSS software). The differences were considered significant when the (901-1065) domain is shown in  (969-995) mutation is included in the minimal paxillin-binding region of FAK. Because FAT recruits FAK to adhesions by an interaction with paxillin and talin, we therefore expected that Del33 mutation would prevent the recruitment of this mutant to FAs. To test this hypothesis, we examined the cellular location of GFP-tagged FAK in epithelial tumor cells MDA-MB-468. In GFP-FAK-WT transfected cells, confocal analysis revealed that GFP fluorescence was co-localized with paxillin . An illustration of the 3D structure of the FAT paxillin , GFP-FAKCells expressing inducible HA-FAK-WT or HA-FAK-Del33 were generated as described in the + transient transfection system. We used the following cell lines: breast cancer cell lines MDA-MB-468, MDA-MB-435s, MDA-MB-231, and MCF7; liver cancer cell lines HepG2 and Hep3B; and normal cell lines REF, Cos7, and NIH3T3. The results shows that Y397 phosphorylation increased when FAK-Del33 was expressed in Hep3B, MDA-MB-468 and MDA-MB-435s cells, and similar tyrosine phosphorylation levels were maintained when FAK-Del33 was expressed in the other cell lines  is achieved by two different mechanisms: 1) following integrin or growth factor receptor activation, Src binds auto-phosphorylated Y397, and the FAK-Src complex further phosphorylated additional Tyr residues within FAK To determine whether FAK-Del33-induced phosphorylation resulted from auto- or trans-phosphorylation, we constructed the kinase-dead mutant K454R as described in the FAK is phosphorylated upon induction by integrin-mediated signaling, such as adherence to fibronectin- or ColI-coated plates, and this phosphorylation is abolished when cells are grown in suspension In addition, cells were trypsinized and suspended in DMEM with 5% FBS for various time intervals. As expected, FAK-WT phosphorylation was reduced over time. However, FAK-Del33-overexpressing cells maintained increased levels of phosphotyrosine, even after incubation for 1 h at 37°C in suspension . This reThe tyrosine kinase Src is thought to play an integral role in regulating FAK phosphorylation. To clarify the role of Src in FAK-Del33 phosphorylation, we transiently transfected FAK-WT, FAK-Del33, FAK-WT/Δ375, and FAK-Del33/Δ375 truncation mutants into MDA-MB-468 cells. The cells were replated onto FN in the presence of the Src inhibitor PP2 (40 µM) or the control compound PP3 (40 µM). Similar to previous research To determine the contribution of an intermolecular mechanism in the auto-phosphorylation of FAK-Del33, we constructed site point mutated (Y397F), kinase-dead (K454R), and a double mutant (Y397F/K454R) in the FAK-WT and FAK-Del33 backgrounds. The same quantity of N.C (plasmids containing FAK-Del33 without further mutation), Y397F, K454R, or both plasmids (Y397F+K454R) was transfected in MDA-MB-468 cells with the FAK-Del33 background . CompareTo determine whether auto-phosphorylation of FAK-Del33 is intermolecular, we also transfected MDA-MB-468 cells with FAK and increasing amounts of the Y397F/K454R double mutant. If FAK auto-phosphorylation occurs through an intermolecular reaction in intact cells, the expression of the Y397F/K454R double mutant will compete in the intermolecular auto-phosphorylation reaction. As expected, we observed a dose-dependent inhibition of Y397 phosphorylation on FAK-WT. However, Y397 phosphorylation of FAK-Del33 was minimally affected . This reTo determine whether auto-phosphorylation of FAK-Del33 can be intramolecular, we depleted the FERM to remove FERM-Kinase intramolecular autoinhibition. We transiently transfected FAK-WT, FAK-Del33, FAK-WT/Δ375, and FAK-Del33/Δ375 truncation mutants into MDA-MB-468 cells. As expected, after FERM domain truncation, Y397 phosphorylation of FAK-WT/Δ375 increased sharply . The cell lysates were subjected to immunoblotting with an anti-Y397 antibody to determine the cellular tyrosine phosphorylation levels. As shown in In this study, we provide interesting insight into the possible mechanism of Y397 phosphorylation in a FAT domain-defective mutant (FAK-Del33). Similar to FAK-WT, Y397 auto-phosphorylation was reduced due to the catalytic domain inactive mutation. In contrast to FAK-WT, FAK-Del33 was constitutively phosphorylated and insensitive to adhesion signals, and Src-mediated regulation of FAK phosphorylation was clearly reduced. This result indicates that FAK-Del33 undergoes auto-phosphorylation, and this event is likely not achieved by integrin stimulation or Src-dependent auto-phosphorylation. Thus, other mechanisms may be involved.A similar mutation with a deletion between residues 965-1012  has been described by Shen Y in vitro with enhanced biochemical signaling and cell motility The mechanism of FAK auto-phosphorylation differs markedly from alternatively spliced isoforms Two FAK auto-phosphorylation models currently prevail. One was proposed by Lietha For FAK-WT, we believe that the two FAK phosphorylation models may be mixed as Y397 phosphorylation can occur both in cis and in Trans. However, for FAK-Del33, which shows decreased intermolecular interactions , we propWork by Lietha et al. has shown that a FAK constructs which lacks the C-terminal region (and hence FAT) is capable of autophosphorylating in cis and trans In conclusion, our paper provides an explanation for why del33, which targets structure and function of the FAT domain, allows autophosphorylation in cis.Table S1Primers used in this paper.(DOCX)Click here for additional data file.


Lactobacillus species dominate the vaginal microbiota of healthy reproductive-age women and protect the genitourinary tract from the attack of several infectious agents. Chlamydia trachomatis, a leading cause of sexually transmitted disease worldwide, can induce severe sequelae, i.e. pelvic inflammatory disease, infertility and ectopic pregnancy. In the present study we investigated the interference of Lactobacillus crispatus, L. gasseri and L. vaginalis, known to be dominant species in the vaginal microbiome, with the infection process of C. trachomatis. Lactobacilli exerted a strong inhibitory effect on Chlamydia infectivity mainly through the action of secreted metabolites in a concentration/pH dependent mode. Short contact times were the most effective in the inhibition, suggesting a protective role of lactobacilli in the early steps of Chlamydia infection. The best anti-Chlamydia profile was shown by L. crispatus species. In order to delineate metabolic profiles related to anti-Chlamydia activity, Lactobacillus supernatants were analysed by 1H-NMR. Production of lactate and acidification of the vaginal environment seemed to be crucial for the activity, in addition to the consumption of the carbonate source represented by glucose. The main conclusion of this study is that high concentrations of L. crispatus inhibit infectivity of C. trachomatis in vitro. Lactobacillus species that dominate the vaginal niche of healthy women9The health and functioning of the female urogenital tract largely rely on its microbial inhabitants23456Extending the concept of lactobacilli as endogenous defence factors, there is an increasing interest for probiotics in the context of urogenital healthChlamydia trachomatis is a leading cause of sexually transmitted infections (STIs) with more than 100 million new cases per year according to global estimates16171819. C. trachomatis has a unique cycle of development, alternating between two distinct bacterial forms. The elementary body (EB) is infectious but non-dividing. In contrast, the reticulate body (RB) is non-infectious but replicativeC. trachomatis, around 18 h post-infection, RBs resulting by binary fission differentiate back into EBs that will afterwards be expelled from the cell, either by exocytosis or cellular lysis 48–72 h post-infectionThe obligate intracellular bacterium Lactobacillus and C. trachomatis in the vaginal environment have not yet been elucidated.In spite of the importance of a healthy vaginal microbiota in preventing genital infections, only a few studies have focused on the protective effects of vaginal lactobacilli towards chlamydial infection2223Lactobacillus strains isolated from the vagina of healthy women on infectivity of C. trachomatis EBs against HeLa cells, as well as to identify metabolic profiles related to the antibacterial activity. The identification of Lactobacillus strains and/or metabolites active against C. trachomatis is the first step of a broader research project aimed at identifying new probiotic strategies to prevent a sexually transmitted infection that adversely affects women’s health.The aim of the present work was to investigate the impact of seventeen C. trachomatis, we evaluated the ability of seventeen Lactobacillus strains to inactivate infectivity of EBs, before they interact with cellular host receptors. These lactobacilli were previously isolated from vaginal swabs of healthy premenopausal womenLactobacillus crispatus (BC1-BC8), Lactobacillus gasseri (BC9-BC14) and Lactobacillus vaginalis (BC15-BC17)  .Lactobacillus CFS, the capacity of C. trachomatis EBs to infect HeLa cells was assessed by immunofluorescence. EBs infectivity was expressed in terms of percentage of inclusions forming units (IFU)/field (median  ±  median absolute deviation) compared to control  of lactobacilli cell free supernatants (CFS) were tested at three different time points . After the challenge with  control .Lactobacillus strains significantly reduced the infectivity of C. trachomatis EBs against HeLa cells. Ten Lactobacillus strains  completely abolished the infectivity of Chlamydia EBs at any time point. The supernatants of five Lactobacillus strains  decreased C. trachomatis infectivity at any contact time, with a complete inhibition after a long term exposure (60 min). L. vaginalis BC17 showed a moderate anti-Chlamydia activity at short contact time (7 min) and L. gasseri BC10 did not exert any inhibitory activity . Lactobacillus gasseri BC10 and BC14, and L. vaginalis BC15 CFS did not alter Chlamydia EBs  decreased C. trachomatis infectivity when applied for short contact times, while no Lactobacillus strain was effective after 60 minutes of exposure. At the lowest concentration, L. crispatus BC1 and BC7, and L. gasseri BC12 supernatants did not exert any inhibitory activity against C. trachomatis EBs (At the highest supernatant concentration (dilution 1:1), the majority of activity . L. crisydia EBs . At the atis EBs .L. crispatus supernatants were the most powerful in counteracting C. trachomatis infectivity, as all of them abrogated Chlamydia inclusions at the highest concentration (1:1), and retained the greatest reductions at the intermediate (1:10) and lowest (1:100) concentrations, especially BC6 and BC8 strains. Six strains  out of eight belonging to L. crispatus species maintained a good activity even at 1:100 concentration; indeed, they caused a significant reduction of Chlamydia infectivity for at least one time point. Among L. gasseri and L. vaginalis strains, heterogeneous activity profiles have been found, especially at the highest concentration: L. gasseri BC9 and L. vaginalis BC15 and BC16 supernatants were very effective in C. trachomatis inhibition, in contrast to L. gasseri BC10 and L. vaginalis BC17.In general, C. trachomatis was strictly concentration-dependent, being fifteen CFS (out of seventeen) effective in reducing EBs infectivity at the highest concentration, whereas only eleven retained a certain efficacy after 1:100 dilution. Notably, at the highest concentration, lactobacilli culture supernatants were found to have pH values comprised in the range 3.71-5.28 . On the contrary, diluted lactobacilli supernatants showed higher pH values, in the range 4.3–7.15  for the dilution 1:10, and in the range 6.76–7.31  for the dilution 1:100 . Moreover, both at the intermediate and lower concentration lactobacilli CFS exhibited higher efficacy when applied for short contact times (7 and/or 15 min).The inhibitory activity of lactobacilli supernatants towards on 1:100 . This fi8, 2.5 × 107 and 2.5 × 106 CFU/mL) and time points . The results are expressed in terms of percentage of C. trachomatis IFU/field (median ± median absolute deviation) compared to control  were tested at three different concentrations  strongly reduced C. trachomatis infectivity at any time point. For short contact times (7 and/or 15 min) a significant inhibitory activity was observed for ten lactobacilli . L. crispatus BC2 and L. gasseri BC14 cells did not affect C. trachomatis EBs infectivity (7 CFU/mL), only L. crispatus BC3 retained a strong inhibitory activity at any time point. L. crispatus BC1, BC2, BC4, BC6, BC8 and L. gasseri BC9, BC11, BC13, and L. vaginalis BC15 significantly reduced C. trachomatis infectivity at short exposure times, while L. crispatus BC7 showed activity after 60 minutes of contact. No inhibitory effect was exerted by L. crispatus BC5, L. gasseri BC10, BC12, BC14, and L. vaginalis BC16 and BC17  and for all L. vaginalis strains. Moreover, the challenge experiments with lactobacilli cells confirmed the major efficacy for short contact times (7 and/or 15 min), independently of the cell concentration.In analogy with the results obtained using the supernatants, C. trachomatis infectivity was evaluated after challenge of EBs for 7, 15 and 60 min. Two concentrations of lactic acid (10 mM and 50 mM), corresponding to the mean titer and the highest titer registered by 1H-NMR in Lactobacillus CFS  to compare the effects of lactic acid with those exerted by an inorganic acid. The results are expressed in terms of percentage of Chlamydia IFU/field (median ± median absolute deviation) compared to control , lactic acid was able to strongly inhibit EBs infectivity, both at 10 and 50 mM, and for all exposure times. After 60 min of contact, act time . HCl didpH value . These rLactobacillus strains on the basis of their capability to counteract Chlamydia infectivity, we approached a statistical analysis on the entire set of median values, considering any concentration and time point. As a first step, we wondered if lactobacilli CP and CFS fractions were equally effective in reducing Chlamydia infectivity. To address this question, we firstly compared all median data collected with Chlamydia EBs pre-incubated with CP fractions to data of untreated EBs, by means of a non-parametric statistical test: the infectivity of Chlamydia EBs pre-incubated with CP was not significantly different from the infectivity of untreated EBs . Similarly, we compared median data obtained with Chlamydia EBs pre-incubated with CFS fractions to data of untreated EBs, and we ascertained that pre-incubation of EBs with CFS significantly reduced Chlamydia infectivity . Indeed, comparing median data obtained with CP-treated EBs to those obtained with CFS-treated EBs, we confirmed that anti-Chlamydia effect of lactobacilli CFS were significantly different from that of the respective CP .With the aim to delineate a ranking of Chlamydia infectivity, we classified lactobacilli only on the basis of the anti-Chlamydia activity exerted by CFS. For each Lactobacillus strain, CFS efficacy has been expressed as the odds between data collected with CFS-treated EBs and untreated EBs , by means of the 1-tailed Wilcoxon signed rank P-values  consists of lactobacilli with P-values ranging between 0.2 and 0.6; the last group  comprises lactobacilli with P-values over 0.6, i.e. lactobacilli that are less effective toward Chlamydia infectivity. Group H counts 7 strains , group I comprises 5 strains , group L included 5 strains as well .Being CFS the fraction capable of reducing P-values . A low Pactivity . The fir1H-NMR to delineate their metabolic profiles. We identified 40 molecules mainly belonging to the families of aminoacids, organic acids monosaccharides, ketones and alcohols.Lactobacilli CFS were analyzed by Chlamydia activity and metabolome of latobacilli  1 and PC2 accounted for the 44.3% of the whole variance of the investigated samples , created on the basis of their inhibitory activity against Chlamydia EBs, in relation to the metabolome  model was built on the concentrations of the identified molecules in order to search for correlations between anti-obacilli . In the  samples . The firtabolome . Strainset al.Chlamydia (group H) while phenylalanine, isoleucine, valine and tyrosine were produced in greater concentrations by lactobacilli less effective against Chlamydia (group L); glucose was consumed at higher levels by lactobacilli belonging to group H while tryptophan was consumed more by lactobacilli of group L. Orotate and phenylalanine production was found to be statistically different between lactobacilli in groups H and L . Inhibitory activity of orotic acid was evaluated, but no significant reduction of C. trachomatis infectivity was observed at the highest concentration found in the lactobacilli supernatants (30 μM) . We added glucose at the concentration of 30 mM in order to reset the sugar consumption by these active lactobacilli. The addition of glucose to L. crispatus BC1 supernatant led to a significant increase (51 fold) of C. trachomatis infectivity at the shortest contact time (7 minutes), while no increase of infectivity was found at the time points 15 and 60 minutes. Similarly, L. gasseri BC13 supernatant enriched with glucose showed a reduction in anti-Chlamydia activity both after 7 minutes (infectivity increase: 8.7 fold) and 15 minutes (infectivity increase: 6.1 fold) of contact  . Orotic  (30 μM) . However contact . These dChlamydia trachomatis is the most common cause of bacterial sexually transmitted infections in both industrialized and developing countries15Lactobacillus species, and the incidence of sexually transmitted infections, including C. trachomatis6282930in vitro study provides experimental evidence for inhibition of C. trachomatis infection by vaginal lactobacilli. It has been hypothesized that Lactobacillus species play a critical protective role in the vaginal niche by producing lactic acid, responsible for low pH, which inhibits sexually transmitted pathogens. Recently, Gong et al. suggested that low pH is fully responsible for chlamydiacidal activity of lactobacilliThis in vitro study showed that Lactobacillus strains exert a strong inhibitory effect on Chlamydia infectivity mainly through the action of metabolites secreted out of the cell, in a concentration-dependent manner. Because organic acids are the principal metabolic products of lactobacilli, the factor “concentration” is inversely correlated to the pH of the culture medium. Indeed, cell-free supernatants corresponding to the highest lactobacilli concentration were characterized by low pH, while diluted lactobacilli supernatants showed higher pH.The present Chlamydia EBs infectivity when applied for short contact times. The inhibition may be generated by a rapid and dynamic modification of EBs membrane induced by the interaction with lactobacilli; we assume that this modification could revert for longer exposure times. The finding of this in vitro study suggests that lactobacilli could exert their protective role against Chlamydia in the early steps of the infection, probably due to inactivation of EBs before they can colonize and infect the host hepitelial cells, according to results previously describedThe factor “contact time” also seems to play a role in the inhibitory activity. Lactobacilli, especially as regards the cellular fractions, were more effective in inhibiting Lactobacillus strains into three groups  according to their ability to counteract Chlamydia infectivity. The best anti-Chlamydia profile was shown by strains belonging to L. crispatus species. Conversely, L. gasseri and L. vaginalis showed a heterogeneous spectrum of activity against C. trachomatis. A similar correlation between biological activity and taxonomy has recently been reported in relation to the antagonism exerted by the same Lactobacillus strains towards Candida infectionIn order to interpret rationally the wealth of data produced in this study, we used an approach that allowed us to classify L. crispatus promotes the stability of the normal vaginal microbiota32L. crispatus inhibit in vitro the growth of uropathogens and block their adhesion to vaginal epithelial cellsL. crispatus was also shown to reduce the adhesion of Neisseria gonorrhoeaeC. trachomatisL. crispatus to limit the inflammatory reaction in C. trachomatis-infected HeLa cells and macrophagesin vitro studies. Lactic acid is able to inactivate a wide range of reproductive tract pathogens, including C. trachomatisChlamydia activity of lactic acid is merely associated with the pH, or other mechanisms may be involved. Lactic acid, at the concentrations found in the lactobacilli supernatants, was able to strongly inhibit EBs infectivity only at acid pH for all exposure times, differently from hydrochloric acid that did not show any activity in the same experimental conditions. These results indicate that a high concentration of H+ ions is necessary but not sufficient to inhibit Chlamydia EBs. Therefore, the presence of lactate in an acidic environment seems to be crucial for the activity. It remains to elucidate the specific mechanisms by which lactate moiety inactivates chlamydial EBs. Notably, the inhibitory activity exerted by the majority of Lactobacillus supernatants, characterized by similar lactate concentrations and pH values, was higher than that exerted by lactic acid solution, suggesting that other metabolites present in lactobacilli supernatants could determine a synergistic effect. Because L. crispatus strains were found to be the most active in counteracting Chlamydia infection, we assume that the effect of lactic acid may be enhanced by the pool of metabolites especially produced by this species.Directly linked to the presence of lactobacilli, the production of lactic acid is accepted as a hallmark beneficial activity of the vaginal microbiota. Lactic acid has been associated with pathogen exclusion and its concentration could also be seen as an important biomarker of vaginal health, although the current evidence is still mainly based on Chlamydia, we studied by 1H-NMR the profile of metabolites secreted by the vaginal lactobacilli. Metabolic variance was strictly correlated with the inhibitory activity, confirming the excellent anti-chlamydial profile of the majority of L. crispatus strains. By considering the median values of lactobacilli grouped according to anti-Chlamydia activity, major differences in the metabolomes were observed between the strains with low activity compared to the strains with high or intermediate activity, which in turn appeared more similar to each other.Given the importance of the metabolic component in determining the inhibition of Chlamydia. Chlamydial EBs, adapted for extracellular survival and primed for infection of susceptible host cells, have historically been described as metabolically dormant. On the other hand, it has been recently reported that C. trachomatis EBs have the capacity for considerable metabolic and biosynthetic activity and utilize glucose as an energy sourceChlamydia. Notably, an increase of glucose concentration in the vaginal fluids collected from women affected by bacterial vaginosis was reported in a recent investigationInterestingly, the metabolomic analysis highlighted the increased consumption of glucose by the most active strains. We demonstrated that glucose depletion represents an additional mechanism of action for lactobacilli antagonism towards in vitro study demonstrates the ability of different Lactobacillus strains of vaginal origin to inactivate C. trachomatis EBs through the production of extracellular metabolites in an acidic environment. In particular, we found that high concentrations of L. crispatus inhibit C. trachomatis infectivity in vitro, stressing once again the importance of this species for the vaginal health. BecauseC. trachomatis is an intracellular organism, further studies will be needed to investigate the interactions between C. trachomatis-lactobacilli-epithelial cells.In conclusion, the present Lactobacilli were cultured in de Man, Rogosa and Sharpe (MRS) broth  supplemented with 0.05% L-cysteine. Incubation was carried out in anaerobic jars supplemented with Anaerocult C  for 18 h at 37 °C.600) of 0.4 corresponds to a cell concentration of 108 colony forming unit (CFU)/mL. Lactobacilli cultures were adjusted to an OD600 of 2.0 with sterile MRS (cell concentration: 5 × 108 CFU/mL) and centrifuged at 5,000 × g for 10 min at 4 °C. Supernatants were filtered through a 0.2 μm membrane filter to obtain stock cell free supernatants (CFS). Ten-fold dilutions (1:10 and 1:100) of the stock CFS were prepared in sterile saline. Cell pellets (CP) were washed and resuspended in sterile saline to obtain stock suspensions of 5 × 108 CFU/mL.The turbidity of 18-h lactobacilli cultures was measured by a Beckman spectrophotometer , considering that an optical density , supplemented with 10% foetal bovine serum, 1% L-glutamine 200 mM, and antibiotics . For the preparation of EBs, confluent HeLa cells were infected with Chlamydia in DMEM medium supplemented with cycloheximide 1 μg/mL, centrifuged at 640 × g for 2 hours to facilitate cell penetration, then incubated at 37 °C with 5% CO2 for 48 hours8, 2.5 × 107 and 2.5 × 106 CFU/mL). Lactobacilli CFS  and CP  were mixed with 5 × 103 IFU of EBs and diluted to 200 μL with sterile phosphate buffered saline. pH values were measured in the final volume. The same amount of purified EBs was used as control after verifying the lack of effects exerted by MRS medium on EBs infectivity. Mixes were incubated for 7, 15 and 60 minutes at 37 °C in 5% CO2 atmosphere, afterward they were centrifuged at 20,000 × g for 10 minutes at 4 °C. Supernatants were used to infect HeLa cells, grown to confluence in individual tubes containing sterile coverslips, and centrifuged at 640 × g for 2 hours to facilitate cell penetration, then incubated at 37 °C with 5% CO2 for 48 hours. Inhibition experiments were also carried out with lactic acid and hydrochloric acid solutions at different concentrations (10 mM and 50 mM) and pH values (pH 4 and 7), in order to evaluate the effect of pH and organic/inorganic acids to C. trachomatis infectivity. In addition, orotic acid was tested at the concentrations of 30 μM in order to verify the ability of this molecule to inhibit Chlamydia infectivity. Orotic acid 30 μM was also tested in combination with lactic acid , to evaluate a possible synergic effect with lactic acid. In order to address the mechanistic question of the importance of glucose depletion in the inhibition of Chlamydia infectivity, supernatants of L. crispatus BC1 and L. gasseri BC13 (dilution 1:1) were added with glucose 30 mM and used in the inhibition experiments.Inhibition experiments with lactobacilli supernatants were carried out with three dilutions of the stock CSF . Inhibition experiments with lactobacilli cells were carried out with three different CP concentrations , as previously reported2O solution of 3-(trimethylsilyl)-propionic-2,2,3,3-d4 acid sodium salt (TSP) 6.25 mM set to pH 7.0 by means of a 100 mM phosphate buffer. 1H-NMR spectra were recorded at 298 K with an AVANCE III spectrometer  operating at a frequency of 600.13 MHz, following the procedure previously described40Chlamydia activity and metabolome of lactobacilli, a PCA model was built on the concentration changes of the identified molecules, scaled to unit variance. The PCA algorithm calculates and sorts linear combinations of the original variables, so to highlight the data structure by means of a low number of orthogonal projections . The score-plot is the representation of the samples in the generated space and highlights the similarities and differences between the samples. For each molecule, the correlation between the concentration change and the loading value with the component of PCA describing a metabolome-activity link was calculated. When the correlation was higher than 0.6, a statistically significant difference between groups H and L was searched by means of a Wilcoxon test, with an accepted Bonferroni-adjusted P value of 0.05.One ml of CFS obtained from lactobacilli was added to 160 μl of a Dwww.R-project.org), applying the non-parametric signed- or matched paired- Wilcoxon rank tests. 1- or 2-tailed tests were used as specified in the manuscript. Differences were deemed significant for P values < 0.05. Spearman correlation was calculated by using GraphPad software .All statistical analysis were performed by using R computational software .


Bacillus subtilis can differentiate into an endospore; the initiation of sporulation is controlled by the master regulator Spo0A, which is activated by phosphorylation. Spo0A phosphorylation is carried out by a multi-component phosphorelay system. These phosphorylation events on histidine and aspartate residues are labile, highly dynamic and permit a temporal control of the sporulation initiation decision. More recently, another kind of phosphorylation, more stable yet still dynamic, on serine or threonine residues, was proposed to play a role in spore maintenance and spore revival. Kinases that perform these phosphorylation events mainly belong to the Hanks family and could regulate spore dormancy and spore germination. The aim of this mini review is to focus on the regulation of sporulation in B. subtilis by these serine and threonine phosphorylation events and the kinases catalyzing them.Bacteria possess many kinases that catalyze phosphorylation of proteins on diverse amino acids including arginine, cysteine, histidine, aspartate, serine, threonine, and tyrosine. These protein kinases regulate different physiological processes in response to environmental modifications. For example, in response to nutritional stresses, the Gram-positive bacterium  E, σF, σG and σK). In Bacillus subtilis, a landmark of the initiation of sporulation is the activation of the transcriptional master regulator Spo0A. It is activated by phosphorylation through a remarkable multi-component phosphorelay system of autophosphorylating histidine kinases (KinA-KinE)  (B. subtilis (B. subtilis.Many Gram-positive bacteria form endospores in response to stress or nutrient limitation . Spores nA-KinE) . These pnA-KinE) . More renA-KinE) . These pnA-KinE) . They senA-KinE) . In somenA-KinE) . In addiigure 1) . Severalsubtilis . In earlsubtilis . This nesubtilis . Such a subtilis . In thisFigure 1) in order to confer to the spore the resistance properties required to survive extreme conditions of temperature, desiccation and ionization . These two kinases have been indeed shown to participate in the regulation of several mechanisms occurring during the initiation of sporulation   . However, the complete mechanism of regulation, potentially via the kinase activity of PrkA, is not known: how does PrkA act on ScoC? Is it a direct or indirect regulation of ScoC and does PrkA phosphorylate ScoC on Ser/Thr residue(s)? What are the exact targets of PrkA phosphorylation? Though it appears that PrkA is a key player in the regulation of sporulation in B. subtilis, more work needs to be done in order to completely understand the role of this putative STPK. The second regulatory protein YabT is a STPK containing three domains: a transmembrane region, a kinase domain and a DNA-binding domain. YabT kinase activity has been clearly established and targets of YabT have been identified. Binding of YabT to DNA activates its kinase activity; YabT is then able to autophosphorylate and to phosphorylate exogenous substrates  and consistently, RecA was previously identified in a phosphoproteome study revealing the same phosphorylated residue   . Howevern factor . Indeed,bstrates . It coloA mutant . The rec residue . It is,  residue . Similar residue . In vitrell pole . RacA ca SpoIIAA . Interes SpoIIAA . Anotherigure 1)  as well and PrkD . AbrB isand PrkD . It has and PrkD . In addiin vitro on Thr63. In vivo experiments confirmed that YabT is responsible of EF-Tu phosphorylation in the spore since no phosphorylated EF-Tu was found in a yabT mutant   and a low amount of water. However, it has been recently shown that the spore RNA profile is highly dynamic a few days following sporulation . During igure 1) . Moreover so far .B. subtilis can remain dormant for years but return to life quickly after exposure to nutrients or muropeptides  can be divided into three consecutive phases: (i) germination with spore rehydration, release of DPA, cortex hydrolysis, and coat disassembly, then (ii) a ripening period with no morphological changes but a molecular reorganization of the cell, and finally (iii) outgrowth with synthesis of macromolecules, membrane elongation, and cell division . However, these three kinases and possibly other still unknown protein kinases  could contribute to these regulatory mechanisms. PrkC is a transmembrane protein composed of an intracellular catalytic domain and an extracellular regulatory C-terminal region containing several beta-lactam-binding domains. These PASTA domains (for penicillin-binding protein and serine/threonine kinase-associated domains) are predicted to interact with the peptidoglycan (PG)  . Hence, PrkC can phosphorylate several substrates produced during vegetative growth or sporulation and, up to now, more than 10 substrates of PrkC have been identified in vitro. These targets include proteins of carbon metabolism  . Biochemand PrkC . Thus, bigure 1) . The prktabolism  or protetabolism . It has mination . Moreovemination . But, foxykinase . It doesxykinase , 1998. Hxykinase  for the xykinase . Howeverxykinase . In addixykinase . These rxykinase , two-comxykinase .B. subtilis  and several examples highlight their regulatory role in cellular physiology during vegetative growth as well as during sporulation. In this mini review, we focused on their regulatory functions in spores and showed that STPKs have relaxed substrate selectivity that confers to the cell a quick way to adapt to the physiological conditions. Taking into account that cross-phosphorylation events occur among STPKs, BY-kinases, and two-component systems, the regulatory network controlling a spore life is highly dynamic and sophisticated. Given the role of spores in many diseases, understanding mechanisms and regulation of spore formation and spore germination has long been a researcher’s interest in order to find a way to get rid of them more efficiently. However, a new interest has now emerged with the use of spores as a tool in biotechnology (It is now widely accepted that regulatory Ser/Thr phosphorylation is as present in prokaryotes as in eukaryotes and that enzymes responsible for these modifications are mainly eukaryotic-like Ser/Thr kinases. To date, four of these proteins have been characterized in chnology .All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Previous studies have revealed that forkhead transcription factor (FOXO3a) is a critical effector of JNK-mediated tumor suppression. However, it is not clear whether the JNK/FOXO3a pathway is involved in neuronal apoptosis in the developing rat brain after hypoxia-ischemia (HI). In this study, we generated an HI model using postnatal day 7 rats. Fluorescence immunolabeling and Western blot assays were used to detect the distribution and expression of total and phosphorylated JNK and FOXO3a and the pro-apoptotic proteins Bim and CC3. We found that JNK phosphorylation was accompanied by FOXO3a dephosphorylation, which induced FOXO3a translocation into the nucleus, resulting in the upregulation of levels of Bim and CC3 proteins. Furthermore, we found that JNK inhibition by AS601245, a specific JNK inhibitor, significantly increased FOXO3a phosphorylation, which attenuated FOXO3a translocation into the nucleus after HI. Moreover, JNK inhibition downregulated levels of Bim and CC3 proteins, attenuated neuronal apoptosis and reduced brain infarct volume in the developing rat brain. Our findings suggest that the JNK/FOXO3a/Bim pathway is involved in neuronal apoptosis in the developing rat brain after HI. Agents targeting JNK may offer promise for rescuing neurons from HI-induced damage. InhibitMammalian forkhead transcription factor (FOXO) is a critical effector of JNK-mediated tumor inhibition ,6. The FFOXO3a regulation involves multiple pathways, including the pro-survival PI3K/Akt pathway and the pro-apoptotic JNK pathway . JNK reg2/92% N2) was used to produce the HI injury. Sham controls received halothane anesthesia and exposure of the CCA without hypoxia and ligation of the CCA. The rat brains from sham controls and from 0.5, 6, 24, 48 and 72 h after HI were collected for experiments.All animal research was approved by the Sichuan University Committee on Animal Research. Female Sprague–Dawley rats with mixed gender litters were acquired from the animal center of Sichuan University . The mother was provided food and water and housed in a temperature- and light-controlled facility until the pups were 7 days old. For the HI model, we used a previously described method . BrieflyAS601245, a highly specific JNK inhibitor, blocks JNK activity by binding to its ATP-binding site. Pups were anesthetized with 2.5% halothane and intracerebroventricularly infused with vehicle  or 150 nmol of AS601245  dissolved in DMSO into the right cerebral hemisphere 30 minutes prior to HI using a 30-gauge needle with a 5-μL Hamilton syringe (infusion rate 1 μL/min) injections. The AS601245 dose (150 nmol in DMSO) was chosen according to a previous report . The locTo delineate the cellular localization of p-JNK and Bim, double immunolabeling was performed with neuron-specific (NeuN) and astrocytic (GFAP) markers. Paraffin-embedded sections (n = 5) were used for fluorescence double immunolabeling, as described previously . The secThe isolated cortex and hippocampus from the right hemisphere were dissected (n = 5 per group). The samples from the cortex were pooled and processed as described previously . BrieflyJNK activity was measured using a specific kit , and glutathione S-transferase-Jun fusion peptides served as the substrate for JNK, as previously described . In brieBrain injury was evaluated by detecting cell apoptosis as well as calculating infarct volume.Apoptotic cell death was detected using an In Situ Cell Death Detection Kit  according to the manufacturer's instructions. Briefly, sections were deparaffinized in xylene, rehydrated through graded ethanol, treated with 0.1 M citrate solution and 3% hydrogen peroxide at room temperature for 10 min and then treated with proteinase K (20 μg/mL) for 7 min. Sections were washed in PBS and incubated at 37°C for 1 h with Fluorescein-FragEL Labeling TdT enzyme. The total cell populations were stained with DAPI. As negative controls, alternate sections were processed in parallel without TdT enzyme.Brain infarct volume was measured through triphenyltetrazolium chloride (TTC) staining with the method described by Liu et al . BrieflyThe data are represented as the mean±standard deviation (SD). ANOVAs with Bonferroni/Dunnett post hoc tests were performed for multiple comparisons. Significant differences between the two groups were compared using t tests. P<0.05 was considered significant.To quantify p-JNK (active JNK) and total JNK expression after HI in this model, we measured p-JNK and total JNK protein expression levels through Western blot analysis. Total protein was isolated from sham controls as well as from rats at 0.5, 6, 24, 48 and 72 h after HI . We found that the total JNK protein was not obviously changed at the different time points compared with sham controls . HoweverBecause p-JNK is highly expressed at 48 h after HI, double immunofluorescence labeling was performed to determine the cellular localization of p-JNK at 48 h after HI (n = 5). Double staining with the neuronal-specific marker NeuN and the astrocyte-specific marker GFAP showed that p-JNK-immunoreactive cells mainly colocalized with neurons  and astrBecause FOXO3a has been identified as a principal substrate of JNK in tumor inhibition ,6, we quFOXO3a dephosphorylation has been reported to promote FOXO3a translocation into the nucleus in cultured cerebellar granule neurons deprived of growth factors , 13. We To determine whether Bim, a downstream target gene of FOXO3a, was regulated by FOXO3a translocation into the nucleus, we quantified Bim protein expression using Western blot analysis. We found that Bim protein was obviously induced at 0.5 h, peaked at 6 h, returned to baseline at 24 h, and then increased again at 48 h, which lasted through to 72 h . There wCleaved caspase-3 (CC3) is a key executor of the apoptosis cascade reaction. We quantified CC3 protein expression through Western blot analysis. We found that CC3 protein was obviously induced at 6 h, peaked at 24 and 48 h, and remained at a high level at 72 h . There wDouble immunofluorescence labeling was performed to determine the cellular localization of Bim after HI. Because Bim is highly expressed 48 h after HI, paraffin-embedded sections at 48 h after HI were used (n = 5). As shown in To further determine whether JNK was involved in FOXO3a regulation after HI, we utilized AS601245, a highly specific JNK inhibitor, which blocked JNK activity by binding to its ATP binding site . In thisWe next investigated whether AS601245 pretreatment affected FOXO3a expression and phosphorylation. We found that AS601245 pretreatment significantly increased the expression of p-FOXO3a at 48 h after HI compared with the DMSO controls . After nBecause AS601245 pretreatment can rescue the decrease of p-FOXO3a after HI, we further investigated whether JNK was involved in the regulation of FOXO3a translocation from the cytoplasm to the nucleus. We found that FOXO3a was upregulated in the nucleus but downregulated in the cytoplasm 48 h after HI . HoweverThere is crosstalk between the pro-apoptotic JNK pathway and the pro-survival Akt pathway at multiple levels ,28. To fTo further understand whether JNK inhibition could regulate a pro-apoptotic protein Bim, one of the FOXO3a target genes, and CC3 in this model, we used Western blot assays to detect the expression of Bim and CC3 protein after JNK was blocked with AS601245. We found that the Bim and CC3 protein levels were significantly reduced at 48 h in the AS601245-treated cortex compared with the DMSO-treated cortex . After nBecause the blockade of JNK activity by AS601245 decreased the expression of Bim and CC3, we investigated whether cellular apoptosis could be blocked by the downregulation of Bim and CC3. Brain sections from sham controls and 0.5, 6, 24, 48 and 72 h after HI as well as from AS601245-treated sections were examined for cellular apoptosis using TUNEL staining . We found that TUNEL-positive cells were not detected in the sham controls  as well In this study, we first showed that the JNK/FOXO3a/Bim pathway was involved in neuronal apoptosis in the developing rat brain after HI. First, we found that JNK phosphorylation and the reduction of p-FOXO3a preceded neuronal apoptosis, suggesting that JNK phosphorylation and FOXO3a dephosphorylation were involved in mediating HI-induced brain damage. Second, FOXO3a dephosphorylation induced FOXO3a translocation from the cytoplasm into the nucleus, which upregulated levels of pro-apoptosis proteins, such as Bim and CC3. Third, pretreatment with AS601245, a highly specific JNK inhibitor, clearly increased FOXO3a phosphorylation, leading to a decrease in FOXO3a translocation from the cytoplasm to the nucleus and levels of Bim and CC3 proteins. Finally, JNK inhibition with AS601245 reduced neuronal apoptosis and brain infarct volume after HI.JNK is an important stress-responsive kinase that is activated by various forms of brain insults. The activated form of JNK is p-JNK. After phosphorylation, activated JNK can activate its downstream transcriptional factors, triggering the expression of pro-apoptotic target gene Bim, and inducing cell apoptosis . In addiPrevious studies have revealed that JNK regulates the FOXO3a activity at different levels and in multiple modes ,15. PhosContrary to the pro-apoptotic JNK pathway, Akt inhibits cellular apoptosis through the negative regulation of FOXO3a activities ,31. TherBecause FOXO3a is downstream of Akt , we quesThe transcription factor FOXO3a can activate Bim gene expression and promote caspase-independent apoptosis . In addiIn summary, we have shown that the JNK/FOXO3a/Bim pathway is involved in neuronal apoptosis in the developing rat brain after HI, and JNK’s regulation of FOXO3a may be independent of Akt. Agents targeting JNK might provide insight into the protective mechanisms involved in neonatal HI.


Entamoeba histolytica. We have recently demonstrated that an atypical kinase EhAK1 is involved in phagocytosis in this parasite. It is recruited to the phagocytic cups through interaction with EhCaBP1. EhAK1 manipulates actin dynamics by multiple mechanisms including phosphorylation of G-actin. Biochemical analysis showed that EhAK1 is a serine/threonine kinase with broad ion specificity and undergoes multiple trans-autophosphorylation. Three autophosphorylation sites were identified by mass spectrometry. Out of these Thr279 appears to be involved in both autophosphorylation as well as substrate phosphorylation. Over expression of the mutant Thr279A inhibited erythrophagocytosis showing dominant negative phenotype. Multiple alignments of different kinases including alpha kinases displayed conserved binding sites that are thought to be important for function of the protein. Mutation studies demonstrated the importance of some of these binding sites in kinase activity. Binding studies with fluorescent-ATP analogs supported our prediction regarding ATP binding site based on sequence alignment. In conclusion, EhAK1 has multiple regulatory features and enrichment of EhAK1 at the site of phagocytosis stimulates trans-autophosphorylation reaction that increases kinase activity resulting in enhanced actin dynamics and phagocytosis. Some of the properties of EhAK1 are similar to that seen in alpha kinases.Phagocytosis plays a key role in survival and pathogenicity of  Entamoeba histolytica is not yet clear though a few of the molecules that participate in the process has been identified123E. histolytica phagocytosis and investigated the mechanisms by which these molecules participate in phagocytic process using uptake of red blood cells (RBC) as a system56Detailed molecular mechanism of phagocytosis by the protist parasite 2+ and the binding is through the kinase domain. However, the binding does not require active kinase domain. Therefore the kinase dead mutant can also bind EhCaBP1. However, progression of phagocytic cups to phagosomes require active kinase domain as over expression of inactive kinase domain of EhAK1 displayed dominant negative phenotype with respect to phagocytosisE. histolytica phagocytosis.EhAK1 was identified as one of the EhCaBP1 binding proteins by affinity chromatography and mass spectrometryOur results show that EhAK1 displays multiple trans-autophosphorylation needed for optimal and unique activity of the kinase. Different autophosphorylation sites appear to have different roles in EhAK1 activity. Our data suggest that phosphorylation of Thr279 is likely to have a major role in the function of EhAK1.in vitro using histone type (IIIS) and myelin basic protein (MBP) as substrates. Phosphorylation was observed with both wild type full length protein (His-EhAK1) and the kinase domain (GST-KD) but not with the mutant protein (K85A)   and S1. 5A  and Sn (K85A) . Therefon (K85A) . No bandn (K85A) . These r2+ ions (0.5 mM MgCl2)   and S3B. and Ni2+. Km and  and Ni2+. These vnd Ni2+10 confirmi2+ and has kinase regulatory roleEhAK1 carries other conserved sites based on multiple alignments shown in in vitro by fluorescence spectroscopy using TNP-ATP, an analog of ATP. The three conserved key residues Lys 85, Asp 223 and Arg 69 needed for kinase activity were mutated to alanine by site-directed mutagenesis. Tryptophan fluorescence was used for monitoring conformational changes in EhAK1. ATP binding reduced tryptophan fluorescence of the protein similar to that seen for EhC2PKA probable ATP binding site in EhAk1 was deduced from sequence alignment 8. The siATP binding was also monitored by using fluorescent ATP analogue TNP-ATP -adenosine triphosphate) by competition assay. TNP-ATP displays high degree of stimulation in fluorescence emission upon binding to proteins as compared to only buffer; absolute magnitude is dependent on the specific protein environment32P-ATP with purified proteins. The wild type, K85A, D223A and R69A were incubated with γ-32P-ATP and irradiated with UV (254 nm) in order to cross-link proteins with radiolabeled ATP. While cross-linking of γ-32P-ATP with the wild type and D273A mutant was observed (radioactive band in autoradiogram), only low level of cross-linking was visible in the case of R69 and none when K85 mutant was used ] mutants. The purified mutant proteins were tested for autophosphorylation, and the results are shown in Autophosphorylation of the kinase domain can be either through a unimolecular cis-reaction or a bimolecular trans-reaction. To check this, phosphorylation was carried out with GST-tagged EhAK1 (donor) and His-tagged kinase dead mutant K85A (acceptor) . Since oE. histolytica cells. However, the process was delayed in cells over expressing autophosphorylation-defective mutants (T279A) because no phagocytic cups were observed after 3 min of incubation despite attachment of RBCs to amoebic cells  was used for detecting phosphorylated form of EhAK1. The specificity of the antibody was determined by probing purified recombinant protein, phosphorylated protein, T279A, and dephosphorylated protein  and MBP. Our results suggest that EhAK1 is a Ser/Thr kinase. However, it is not inhibited by staurosporine confirming atypical nature of this kinase. Low Km (28 ± 2 nM) for ATP suggests that EhAK1 can work optimally at low ATP concentration once the enzyme is activated. This is physiologically relevant as the parasite may encounter unfavourable nutrient conditions where an enzyme working at low ATP concentrations will help the parasite to endocytose nutrients and aid in its survival.Specificity of many kinases, in terms of the site of autophosphorylation and substrate phosphorylation, has been shown to vary depending on cation used. For example, the kinase domain of EGFROur results suggest that autophosphorylation has an important role in the regulation of EhAK1 activity and consequently, may be a key step in its function. The three autophosphorylation sites are likely to have different functions, Thr 279 for substrate phosphorylation and Ser 54/Ser 398 for autophosphorylation. Since autophosphorylation can enhance rate of substrate phosphorylation, by phosphorylating a combination of sites it is likely that phosphorylation of different substrates can be manipulated. This is particularly relevant as EhAK1 carries out autophosphorylation by a trans-bimolecular reaction. Since over-expression of inactive kinase led to a dominant negative phenotype it can be inferred that an active kinase is required for phagocytosis. Many protein kinases require phosphorylation for full activity, and this modification can be a critical event in the activation of these enzymes1719EhAK1 does show similarity to alpha kinases, a group of atypical kinases that have propensity to use alpha helical regions as substrates. Some of the, these properties are: 1) no strict requirement of divalent cation, lack of sensitivity to ser/thr kinase inhibitor staurosporine, multiple autophosphorylation sites and presence of some sequence features and conserved residues. However, it also displays a few properties, such as phosphorylation of non helical region of actin not known to be a substrate of alpha kinases and presence of SH3 domain. Therefore it is difficult to conclude at present if EhAK1 is indeed an alpha kinase.E. histolytica that have been characterized in detail. This will help in elucidating molecular mechanisms of phagocytosis where EhAK1 is found to participate.In conclusion we have biochemically and functionally characterized EhAK1, an atypical protein kinase with some similarity to alpha kinases. The proposed model that shows the suggested involvement of EhAK1 in amoebic phagocytosis is shown in 32P-ATP) into the band which co-migrated at with purified recombinant protein. We have already seen that the phosphorylated and non phosphorylated forms of EhAK1 co-migrate in SDS-PAGE. The standard reaction mixture  contained 0.5 mM MgCl2, 30 mM HEPES (pH 7.5), protease inhibitor, phosphatase inhibitor cocktail and pure kinase (2 μg). Reactions were initiated by the addition of (γ-32P-ATP) (6000 Ci/mmol) to a final concentration of 2.5 μM and incubated at 30 °C for 1 h and was stopped by adding SDS sample buffer containing 50 mM EDTA followed by boiling. The samples were than resolved on SDS-PAGE. Phosphorylation was detected by a Phosphor Imager (Fujifilm).Autophosphorylation of EhAK1 was measured as the amount of radioactivity incorporated i.e. . Briefly E. histolytica cells were harvested via centrifugation and washed with PBS and re-suspended in TYI-33 medium. The cells were then transferred onto acetone-cleaned coverslips placed in a petridish and was allowed to adhere for 10 min at 35.5 °C. The culture medium was removed and the cells were fixed with 3.7% pre-warmed paraformaldehyde for 30 min. After fixation, the cells were permeabilized with 0.1% Triton X-100/PBS for 1 min. The fixed cells were then washed with PBS and quenched for 30 min in PBS containing 50 mM NH4Cl. The coverslips were blocked with 1% BSA/PBS for 30 min, followed by incubation with primary antibody at 37 °C for 1 h. The coverslips were washed thrice with PBS followed by 1% BSA/PBS before incubation with secondary antibody for 30 min at 37 °C. Antibody dilutions used were: anti-EhAK1 at 1:100, anti-GFP at 1:200, anti-rabbit Alexa 488 and 556, anti-mice Alexa556 (Molecular Probes) at 1:300, TRITC-Phalloidin at 1:250. The preparations were further washed with PBS and mounted on a glass slide using DABCO  octane (Sigma) 2.5% in 80% glycerol). The edges of the coverslips were sealed with nail-paint to avoid drying. Confocal images were visualized using an Olympus Fluoview FV1000 laser scanning microscope. The raw images were processed using FV10-ASW 1.7 viewer or Image J software.Immunostaining was carried out as described before  was added and the mixture was incubated at room temperature, in the dark, for 40 min. Enzymatic digestion was carried out by incubating the reaction mixture with trypsin (40 ng/μl of 25 mM NH4HCO3) and incubation was carried out overnight at 37 °C. After digestion, peptide mixtures were acidified to pH 2.8 with formic acid and desalted with minispin C18 columns . Samples were dried under vacuum and resolubilized in 0.1% formic acid and 2% acetonitrile before mass spectrometric analysis.The purified SDS-PAGE protein band was subjected to in-gel trypsin digestion. Briefly, the excised gel was sliced to small pieces, transferred to a sterile siliconized Eppendorf and destained by repeated washing with 50 mM NHThe tryptic peptide samples were separated by reverse-phase chromatography for each via Thermo Scientific Proxeon nano LC using a C18 picofrit analytical column . The HPLC was coupled to an LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific). Peptides were loaded onto the column with Buffer A  and eluted with 60 min linear gradient from 2 to 40% buffer B  followed by 10 min linear gradient from 40% to 80% buffer B and finally 20 min linear gradient from 80% to 90% buffer B. The mass spectra were acquired in the LTQ Orbitrap Velos utilizing Data-Dependent Decision Tree (DDDT) method (Thermo scientific Application Note: 30179) with full MS scan (RP 60000) followed by 20 data-dependent MS/MS scans with detection of the fragment ions in the ITMS mode along with supplemental activation. Target values were 1 × 106 for full FT-MS scans and 3 × 104 for IT-MS MSn scans.E. histolytica UniProt database also containing decoy database using a target false discovery rate of 1% for strict and 5% for relaxed conditions. phosphoRS node was used for automated and confident localization of phosphorylation sites within validated peptide sequences.Data analysis was performed using Thermo Proteome Discoverer software suite (1.3.0.339 DBVersion: 78). For the search engine SEQUEST, the peptide precursor mass tolerance was set to 10 ppm, and fragment ion mass tolerance was set to 0.8 Da. Carbamidomethylation on cysteine residues was used as fixed modification, and oxidation of methionine, N-terminal acetylation along with phosphorylation of serine, threonine, and tyrosine was used as variable modifications. Spectra were queried against the http://www.proteomexchange.org) via the PRIDE partner repository with the dataset identifier PXD002691The mass spectrometry proteomics data have been deposited to the open access library of the ProteomeXchange Consortium  via semi dry method and was further processed using standard methods. The antigens were detected with polyclonal antibodies raised in rabbit or mice as indicated  anti-pThreonine  followed by secondary anti-rabbit and anti-mice immunoglobulins conjugated to HRPO . ECL reagents were used for visualization (Millipore). GFP and GST antibodies used were obtained from Molecular probes and Santa Cruz, respectively. The concentration of proteins in a sample was estimated by bicinchoninic acid assay using BSA as a standard.How to cite this article: Shahid Mansuri, M. et al. Autophosphorylation at Thr279 of Entamoeba histolytica atypical kinase EhAK1 is required for activity and regulation of erythrophagocytosis. Sci. Rep.6, 16969; doi: 10.1038/srep16969 (2016).


There is still much unknown regarding the computational role of inhibitory cells in the sensory cortex. While modeling studies could potentially shed light on the critical role played by inhibition in cortical computation, there is a gap between the simplicity of many models of sensory coding and the biological complexity of the inhibitory subpopulation. In particular, many models do not respect that inhibition must be implemented in a separate subpopulation, with those inhibitory interneurons having a diversity of tuning properties and characteristic E/I cell ratios. In this study we demonstrate a computational framework for implementing inhibition in dynamical systems models that better respects these biophysical observations about inhibitory interneurons. The main approach leverages recent work related to decomposing matrices into low-rank and sparse components via convex optimization, and explicitly exploits the fact that models and input statistics often have low-dimensional structure that can be exploited for efficient implementations. While this approach is applicable to a wide range of sensory coding models (including a family of models based on Bayesian inference in a linear generative model), for concreteness we demonstrate the approach on a network implementing sparse coding. We show that the resulting implementation stays faithful to the original coding goals while using inhibitory interneurons that are much more biophysically plausible. Cortical function is a result of coordinated interactions between excitatory and inhibitory neural populations. In previous theoretical models of sensory systems, inhibitory neurons are often ignored or modeled too simplistically to contribute to understanding their role in cortical computation. In biophysical reality, inhibition is implemented with interneurons that have different characteristics from the population of excitatory cells. In this study, we propose a computational approach for including inhibition in theoretical models of neural coding in a way that respects several of these important characteristics, such as the relative number of inhibitory cells and the diversity of their response properties. The main idea is that the significant structure of the sensory world is reflected in very structured models of sensory coding, which can then be exploited in the implementation of the model using modern computational techniques. We demonstrate this approach on one specific model of sensory coding  that has been successful at modeling other aspects of sensory cortex. The diverse inhibitory interneuron population in cortex has been increasingly recognized as an important component in shaping cortical activity . HoweverThough the main ideas likely extend to other areas, for concreteness we will focus the present discussion on the primary visual cortex (V1). In V1, visual information is encoded using a rich interconnected network of excitatory principal cells and inhibitory cells, and different coding functions appear to be implemented by distinct inhibitory populations , 3. ThouInhibitory and excitatory interactions arise from distinct cell types, and synapses from an inhibitory cell cannot have excitatory influences on postsynaptic cells and vice versa ;Excitatory neurons generally outnumber inhibitory interneurons, with E/I ratios recently estimated to be in the range 7:1 to 6:1 ; anThe interneuron population has diverse tuning properties , includiFor theoretical and simulation studies to illuminate the role of inhibition in neural coding, it is imperative that coding models begin to incorporate experimental observations regarding the distinct properties of excitatory cells and inhibitory interneurons. Specifically, to realistically investigate the role of inhibition in neural coding, models should incorporate at least three major properties while staying faithful to the coding rule and other desirable properties :The main contribution of this paper is to demonstrate a systematic computational method for effectively incorporating these biophysical interneuron properties into dynamical systems implementing neural coding models. In our proposed approach we exploit the fact that in many cases of interest, the total required inhibition is highly structured due to the relationship between the coding model and the statistics of the inputs being encoded. Similar to efficient coding hypotheses that postulate compact representations of sensory stimuli, the structure of the sensory statistics and the coding model can also be used to implement the required inhibition with a parsimonious computational structure. Specifically, we propose to reformulate the connectivity matrix to respect Dale’s law and exploit the inhibition structure in a matrix factorization to minimize the number of inhibitory interneurons. Furthermore, we leverage recent results from the applied mathematics community on advanced matrix factorizations to develop an approach that demonstrates the observed diversity of orientation tuning properties in inhibitory interneurons.The end result of this approach is a network implementation that is functionally equivalent to the original model, but which has an interneuron population that better respects the three major biophysical properties ignored by many current coding models. In addition to this primary goal of providing a recipe for including inhibitory interneurons into coding models, this approach also suggests possible functional interpretations of some biophysical properties of the interneuron population. In particular, we propose that while Dale’s law may reflect a physical constraint of individual cells, in contrast the E/I ratio can be viewed as an emergent characteristic of a population implementation that maximizes efficiency by minimizing the number of interneurons and thus maintenance costs. In addition, we demonstrate that the orientation tuning diversity in the inhibitory population can arise from differential connectivity patterns between the excitatory and inhibitory cells.G, where the element [G]m, n describes synaptic strength from the nth neuron to the mth neuron. While G can take many forms, the structure is governed by the coding model and the statistics of the stimuli being encoded.In a recurrent network implementing a neural coding model, each node in the network is generally driven by both exogenous inputs  and lateral connections from other cells in the same network. These lateral connections are often described in terms of a connectivity matrix G arises for a family of commonly-used coding models, we consider the Bayesian inference paradigm that has found increasing support as a framework for studying neural coding [s ∈ ℝN  can be approximately written as a linear superposition of M dictionary elements {ϕi} representing basic visual features :ai}, n represents a noise source, and the N × M matrix Φ consists of one dictionary element on each column. These dictionary elements are often interpreted as the receptive fields (RFs) of a principal cell, such as spiny stellate cells or pyramidal cells.To illustrate how l coding . While ts, the coefficients a in the linear generative model  can be found by maximum a posteriori (MAP) estimation. Assuming Gaussian noise and a prior distribution P(a), the MAP estimate is found by minimizing the negative log of the posterior:λ is a scalar capturing the model SNR. When the prior distribution is log-concave  matrix is given by G = ΦTΦ. G can be interpreted as a recurrent matrix because its off-diagonal terms capture the influence between cell activities. In particular when we assume that the prior is independent, i.e. log P(a) = ∑i log P(ai), as is common in efficient coding models, G captures all the recurrent influence. Note that any dynamical system involving a derivative of an energy function such as G of this form.Given the dictionary Φ and the stimulus l family ), the inG (connecting two cells), there are many implementations that would result in a functionally equivalent coding rule. For example, one of the approaches we will utilize is to model the connectivity between the interneurons and principal cells using a matrix factorization:VT matrix captures the synaptic connections onto a set of interneurons from the principal cells, the U matrix captures the synaptic connections from these interneurons back onto the network of principal cells, and Σ is a diagonal matrix representing the independent gains/sensitivity of each interneuron.While the most obvious implementation of the network would use a single interneuron for each entry of As a concrete relevant example, we will demonstrate the proposed approach in the context of a dynamical system implementing a sparse coding model of V1, where a population of cells encodes a stimulus at a given time using as few active units as possible. The sparse coding model  has been shown to be sufficient to explain the emergence of V1 classical and nonclassical response properties –19, poteI is the identity matrix, u are internal state variables for each node , G = ΦTΦ governs the connectivity between nodes, and Tλ(⋅) is the soft thresholding function. Note that despite not using steepest descent on G = ΦTΦ. In the simulations in this study, the dictionary Φ is pre-adapted to the statistics of the natural scene with a standard unsupervised learning method, resulting in Gabor wavelet-like kernels that resemble V1 classical receptive fields [The sparse coding model can be viewed as a special case of inference in the linear generative model described above withposed in  that useposed in , 32, canposed in , and is posed in , 34, 35.e fields .G, but it is agnostic about the network mechanism that implements these interactions. Specifically, the model as described in [This dynamical system model requires influences between cells that are described by the matrix ribed in  does notThis sparse coding network will serve as a concrete demonstration of the proposed strategy to incorporate more biophysically realistic inhibitory interneurons. The example network we use has 2048 excitatory neurons and has the same parameters as in a previous work  see Mat.G into matrices representing excitatory and inhibitory interactions. Specifically, the recurrent connectivity matrix G can be decomposed into inhibitory and excitatory effects:G+ are the positive elements of the matrix (representing the inhibitory recurrent connections) and G− are the negative elements (representing excitatory recurrent connections).As a first step towards a biologically realistic interneuron population encoding model, we show that Dale’s law can be respected in the model by decomposing the recurrent connectivity matrix GExcite can be implemented by direct synapses between excitatory principal cells, the inhibitory component GInhib requires inhibitory interneurons between the relevant principal cells. To capture these disynaptic connections, we factor the inhibitory matrix into two matrices: GInhib = UVT. For a simple stylized illustration, the network in While GInhib. For a simple concrete example, we can achieve the same encoding as I is the identity and plays the role of U; G+ as defined in VT. The resulting network is shown in M inhibitory neurons  and all inhibitory cells in this implementation have the same orientation tuning properties as the excitatory cells , which  to 50:1 .population of excitatory neurons rather than a single excitatory neuron. As an aside, we note that the reasoning above suggests that the inhibitory population should be overcomplete in systems where these neurons do form the explicit stimulus representation. Indeed, this is proposed in a theory of olfactory bulb encoding where granule cell interneurons form the olfactory representation and are an overcomplete population [In contrast, if inhibitory neurons only need to achieve a computational goal for the circuit without requiring these same stimulus coding properties, there is no need for an overcomplete inhibitory population. In fact, the system could exploit this structure to use the fewest number of inhibitory cells possible to avoid incurring unnecessary cell maintenance costs . In contpulation .G? Said mathematically, what choice of factorization results in the fewest number of inhibitory cells, corresponding to the number of columns of U and V? In many cases of interest, the connectivity matrix G is likely to be low-rank (i.e. M > rank(G)), providing an opportunity to achieve an efficient implementation of the interneuron population by “compressing” the recurrent connectivity to its most essential components. There are two different causes of low-rank structure in G for the types of models considered in this study. First, an overcomplete representation of the principal cells implies directly that G is low-rank  = rank(ΦTΦ) = rank(G)). Second, natural images are highly structured, meaning that image patches have fewer “degrees of freedom” than the number of photoreceptors N being used to transduce the image (i.e. N > rank(Φ) = rank(G)) [G.A natural question to ask is, what is the minimum number of inhibitory cells required to implement the influences specified by the matrix rank(G)) , 44. ThiG as a Gramian matrix gives rise to the following decomposition of the recurrent matrix:M > N), the resulting E/I ratio is M:N and requires  fewer inhibitory cells than excitatory cells. However, this implementation does not produce the diversity of tuning properties observed in V1 interneurons, which can be either orientation tuned or non-orientation tuned (with no apparent structure) [In detail, these two sources of low-rank structure can be exploited to achieve the same coding function of ructure) . In factructure)  of theseG encodes redundant structure in natural scenes, the recurrent connectivity can be represented by an even lower dimensional decomposition than G . Written mathematically, this approximation is:F is the Frobenius norm. This is equivalent to solving:r and ϵ. The solution to this problem can be found by the truncated singular value decomposition (SVD), known commonly as Principal Component Analysis (PCA). Note that in our case the truncated singular values are equivalent to the truncated eigenvalues because G is symmetric semi-positive definite. Specifically, we can decomposeU and V are truncated singular vectors with orthogonal columns and implement the recurrent synaptic weights .The resulting inhibitory population receives dense, low-rank connections from the principal cells with synaptic weights defined by G, representing a significant savings compared to 2048 and 256 interneurons required in Eqs  is included so that the remainder L has a lower rank than G.Recently the applied mathematics community has developed a principled algorithmic approach known as Robust PCA (RPCA) –48 that G = ΦTΦ into a low-rank matrix L and a column-sparse matrix S by solving the following convex optimization program iteratively:* is the nuclear norm  to encourage L to have low rank, ‖⋅‖1 is the ℓ1-norm  to encourage sparsity, and Λ is a diagonal weighting matrix updated at each iteration to encourage column sparsity in S. The update rule for Λ is given bySi), we now model this difference using a structured matrix S.In the context of our study, an unstructured sparse connectivity matrix can result in a relatively large number of interneurons because there can be a large number of columns containing at least one non-zero value. To maintain the small number of interneurons, we also want the sparse matrix to be row or column-sparse  on the low-rank matrix  earlier , the equal cells .With a parameter choice that strikes a balance between sparseness and low rank see , the E/I1 in L, and has properties described in the previous section. The second subpopulation (exemplified by I2 in S. This population receives one-to-one (i.e. sparse) connections with unit weights defined by the diagonal matrix D from the principal cells, and projects back to the principal cells with weights defined by S. Because S is column-sparse, the rows in D that correspond to the zero columns in S can be set to 0 without altering the recurrent influence. Said another way, we can eliminate the zero rows of the D matrix and the zero columns of S, meaning that only a few interneurons are required in this subpopulation (Decomposing the connectivity matrix in this manner results in two distinct populations of inhibitory interneurons with a relative size controlled by the magnitude of the average weights in the matrix Λ. The first subpopulation (exemplified by the inhibitory cell Ipulation .L in the approximation to the recurrent matrix. This is shown in a‖0) and the relatve ℓ2 error for the input image (‖s−Φa‖2/‖s‖2). As demonstrated in This model network accurately solves the sparse coding inference problem , despiteInterestingly, the sparse and low-rank interneuron populations in RPCA show the same kind of diverse orientation tuning as V1 inhibitory cells in vivo. The low-rank inhibitory population has RFs that are mostly untuned . The spaaI, L1(t) and aI, L2(t) are the dynamic responses of the two low rank interneuron populations and aI, S(t) is the response of the sparse population. Here we assume that inhibitory neurons have the same time constant as the principal cells. As shown in For simplicity in the above model we treat the interneurons as instantaneous linear units . To makeThe main contribution of this study is a biologically plausible computational framework for including inhibitory interneurons in efficient dynamical system models of neural coding based on ideas from matrix factorization and convex optimization. From the demonstrated results, we conclude that techniques such as low-rank plus sparse decomposition can be used to find implementations of a recurrent connectivity matrix that produce equivalent population dynamics while using an inhibitory structure that matches many biophysical properties, including respecting Dale’s law, known E/I cell ratios, and diversity of orientation tuning properties. In our example of a network implementing sparse coding, the resulting representation is nearly as accurate as the idealized coding model while being much more faithful to the biophysics of the inhibitory population. Because the proposed approach only depends on the structure of the recurrent matrix , we expOur approach suggests that the excitatory to inhibitory cell ratio in V1 is an emergent property of interneurons implementing efficient visual coding in a resource-conserving way. Specifically, in our model a comparatively small number of interneurons efficiently route the inhibitory influence by taking advantage of the overcomplete and low-rank (redundant) structure in the recurrent connectivity pattern. We have further demonstrated that the tuning diversity of interneurons could arise from differential connectivity with the excitatory population—a prediction that could be tested experimentally.Recently a few studies explicitly introduced inhibitory interneuron populations into high-level functional encoding models. Lochmann et al.  developeIn a more recent work, Boerlin et al.  illustraAnother recent study  has deveOur model gives several experimentally verifiable predictions of interneuron properties that we detail in this section. We also note that while biologically plausible, there are limitations with the current model (see the Caveats section later).First of all, our model predicts the existence of two distinct connectivity patterns between inhibitory interneurons and principal cells: the recurrent connections between principal cells and the low-rank interneurons are dense while the recurrent connections between the principal cells and the sparse interneurons are selective. According to these patterns, a likely biological correlate for the low-rank interneurons in mice is the fast-spiking parvalbumin-expressing (PV) interneurons, which receive dense synaptic inputs from nearby pyramidal cells of diverse selectivities , and proIt is less clear what biological correspondence is most appropriate for the sparse interneuron population arising in the model. One candidate is the irregular firing cannabinoid receptor-expressing (CB1+) neurons, which have been shown to be more sparsely connected to the principal cells than the PV neurons . HoweverIn addition to general connectivity patterns, our model also provides predictions on the distribution of inhibitory synaptic weights in V1. As shown in GInhib term. The excitatory influences are assumed to be implemented by direct excitatory-excitatory connections represented by the connectivity matrix I−GExcite. The identity matrix I is assumed to be implemented by an independent mechanism that results in self-excitation. Biologically, there are at least three ways this self-excitation could be achieved: through “autapses” [In discussing the recurrent connections in the network of utapses”   Lagrangian minimization and a dual update. The outer loop updates Λ according to a in λ = 0.1, as was done previously in [We start with a model network of 2048 principal neurons with receptive fields adapted to 16 × 16 natural image patches using sparse coding . The priously in .L and the number of non-zero columns in S. To achieve a biophysically accurate small E/I cell ratio, we would like both the rank of L and the number of non-zero columns of S to be small. However, these are two competing requirements whose tradeoff depends on the parameters in Eqs  in each of the two low-rank inhibitory populations with a total of 220 cells so that 99% of the variance in L was retained. We also used 220 interneurons in the SVD implementation to facilitate comparison between the models.In the proposed implementation, the required number of inhibitory interneurons is governed by the rank of s in Eqs  and 14)L and theS1 Text(PDF)Click here for additional data file.S1 FigFeedforward push-pull could also be implemented with fewer inhibitory neurons than excitatory neurons.(TIF)Click here for additional data file.S2 FigA) The recurrent network that implements the global inhibition (Eq. (S8)). I1 pools all activities from the excitatory population, weighs them by c, and projects back to the excitatory population. (B) The orientation tuning curve of the inhibitory neuron I1.((TIF)Click here for additional data file.