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No anti-CP5 antibodies were observed for the PFESA0119 capsule knockout mutant ( Table 4 ) . | PMC6331205 | -1 | [] |
CP8-specific cLIA was also used to evaluate immune responses to CP8 isolates that lacked capsule expression in vitro . | PMC6331205 | -1 | [] |
Anti-CP8 antibodies were detected in mice infected with three CC12 isolates ( PFESA1305 , PFESA1405 and PFESA2455 ) and the CC188 isolate ( PFESA2058 ) that harbored mutations in the cap8 operon . | PMC6331205 | -1 | [] |
No immune responses were detected in mice infected with the single CC12-t160 isolate ( PFESA1502 ) that carried the same mutations in cap8D and cap8E , but was CP-negative in vivo ( Table 5 ) . | PMC6331205 | -1 | [] |
These results are consistent with the IFA data and further support the observed in vivo capsule expression for CP8 isolates . | PMC6331205 | -1 | [] |
Discussion . | PMC6331205 | -1 | [] |
Owing to the extended length of vaccine research and development , it is important to monitor the epidemiology of the target pathogen throughout the development program to ensure that the vaccine candidate under development remains appropriate . | PMC6331205 | -1 | [] |
Over recent years , the US has seen a change in the epidemiology of S. aureus as exemplified by the rise of CA-MRSA USA300 isolates and their establishment in healthcare settings [ 9 ] . | PMC6331205 | -1 | [] |
Analysis of these contemporary disease isolates revealed that they carried a mobile methicillin resistance cassette and specific virulence factors such as PVL , and appeared to lack capsular polysaccharides expression in vitro [ 55 , 88 , 90 ] . | PMC6331205 | -1 | [] |
The most advanced vaccine candidate in clinical studies is SA4Ag , a four-antigen vaccine which includes CP5 and CP8 conjugated to the CRM197 carrier protein in addition to two protein antigens . | PMC6331205 | -1 | [] |
The observation that emerging isolates may not express two of the vaccine ’ s target antigens , ( CP5 or CP8 ) highlighted the importance to further investigate the distribution of capsule genotypes in prevalence-based studies and to assess the proportion of isolates that express capsular polysaccharides . | PMC6331205 | -1 | [] |
This study is also the first that carefully and comprehensively examined the clonal relatedness ( CC and spa types ) , CP genotype , and CP phenotype ( in vitro and in vivo ) amongst a panel of relevant S. aureus clinical isolates . | PMC6331205 | -1 | [] |
A prevalence-based collection of such S. aureus isolates in the US was utilized to permit an epidemiological survey in a variety of clinical settings.The S. aureus population circulating in the US during the time periods studied was represented by 13 prominent clonal complexes . | PMC6331205 | -1 | [] |
In concordance with previous reports of S. aureus infection and colonization in the US and Europe , the S. aureus isolates in healthcare and community settings were primarily distributed between four major CC5 , 8, 30 , and 45 [ 91 – 96 ] , with CC8 and CC5 comprising the largest number of isolates . | PMC6331205 | -1 | [] |
In agreement with recent studies showing an association between invasive disease and clonality [ 97 ] , we also observed a significant association between CC and type of infection ( invasive vs non invasive ) ( P = 0.036 ) . | PMC6331205 | -1 | [] |
The inpatient data shows that although most S. aureus genotypes ( CC ) have the capacity to cause invasive disease , isolates within CC5 , 8, 30 and 45 caused the majority of invasive disease . | PMC6331205 | -1 | [] |
In the current study , the most prevalent spa types in the US S. aureus population were t008 and t002 , consistent with results from earlier reports [ 14 ] . | PMC6331205 | -1 | [] |
There was a strong association between spa type and type of infection ( HA-wound vs respiratory or invasive ) ( P < 0.001 ) , t008 isolates caused a higher proportion of wound infections whereas t002 isolates predominated in HA-invasive and HA-respiratory infections.We determined CP prevalence , genotype distribution and expression in S. aureus isolates circulating in the US . | PMC6331205 | -1 | [] |
Most available literature reports described capsule prevalence as determined by serotyping [ 53 , 54 , 56 ] . | PMC6331205 | -1 | [] |
The present study is the first systematic molecular analysis of CP distribution and expression both in vitro and in vivo of S. aureus isolates causing disease in the US during 2004 and 2009 – 10 . | PMC6331205 | -1 | [] |
All S. aureus isolates harbored the genetic pathway to express either CP5 ( 72 % ) or CP8 ( 28 % ) . | PMC6331205 | -1 | [] |
A link was found between capsule genotype and CC-spa type , supporting the previous notion of the high clonality of the capsule antigens [ 67 ] . | PMC6331205 | -1 | [] |
Among the MRSA isolates , 96 % of the isolates were CP5 while 50 % of the MSSA strains were CP5 ( P < 0.001 ) . | PMC6331205 | -1 | [] |
Overall ( MRSA and MSSA isolates combined ) , CP5 genotypes were the most prevalent in both inpatient and outpatient wards ( P < 0.001 ) . | PMC6331205 | -1 | [] |
For a vaccine strategy to be effective , the vaccine must contain antigens that are expressed in host microenvironments and elicit immune responses in humans . | PMC6331205 | -1 | [] |
A mechanism of protection for multicomponent vaccines containing CP conjugates is to induce antibodies that facilitate the killing of the bacteria by complement mediated opsonophagocytosis which can be measured in vitro by OPA . | PMC6331205 | -1 | [] |
We demonstrated in this study that humans immunized with a vaccine ( SA3Ag ) containing CP conjugates generated CP-specific antibodies with bactericidal activity against encapsulated isolates in OPA and that genetic lineage is not linked to the potential for a strain to be killed by anti-CP antibodies . | PMC6331205 | -1 | [] |
CP-specific antibodies were detected in mice infected with two S. aureus USA300 isolates and not in mice infected with a USA300 capsule knockout mutant , indicating in vivo expression of capsule in these isolates ( Table 5 ) . | PMC6331205 | -1 | [] |
Potential vaccine candidate antigens must be present in the genome of circulating isolates , highly conserved , and expressed by S. aureus during the course of natural infection . | PMC6331205 | -1 | [] |
The danger in relying on in vitro assays to preselect antigens is the potential lack of expression using standard culture conditions . | PMC6331205 | -1 | [] |
Several studies comparing S. aureus gene expression in vivo to that under in vitro growth conditions indicated that the behavior of S. aureus in vivo could be significantly different from that observed in vitro [ 98 – 101 ] . | PMC6331205 | -1 | [] |
Some candidate antigens are expressed both in vivo and in vitro ( e.g . | PMC6331205 | -1 | [] |
clumping factor A [ 21 , 102 , 103 ] ) , while others are poorly expressed or not detectable in in vitro grown bacteria [ 59 , 104 , 105 ] . | PMC6331205 | -1 | [] |
Manganese transporter protein C ( MntC ) , a protein that is not readily detected using standard culture conditions , was identified as a potential vaccine target based on in vivo expression analysis showing that MntC is rapidly upregulated by S. aureus in a murine model of infection [ 106 ] . | PMC6331205 | -1 | [] |
In this study , assessing S. aureus isolates for capsule production revealed that a proportion of isolates tested within CC8 ( 37 % ) did not express capsule in vitro . | PMC6331205 | -1 | [] |
Likewise , a lack of in vitro capsule expression was observed for other isolates belonging to CC5 , 12 , 97 and 188 , albeit to a lesser extent . | PMC6331205 | -1 | [] |
In vivo capsule expression analysis of representative isolates that showed no capsule expression in vitro revealed that many of these isolates were perfectly capable of expressing capsule in vivo . | PMC6331205 | -1 | [] |
We therefore examined the cap operons of all isolates in the collection to identify whether any mutations within the cap operons could be linked to an in vitro CP-negative phenotype.Both Cocchiaro et al. and Boyle-Vavra et al. [ 51 , 55 ] identified specific genetic mutations in the capsular polysaccharide biosynthetic operon , which they showed by complementation experiments may contribute to the lack of observed capsule expression in vitro . | PMC6331205 | 20,568 | [
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In an effort to assess the relevance of these mutations with regard to capsule expression during infection , Boyle-Vavra et al. [ 55 ] conducted an in vivo analysis using a single isolate and confirmed that no capsule expression could be detected in that instance . | PMC6331205 | -1 | [] |
The methodology used was different from that utilized in our previous reports where expression was detected [ 21 , 64 ] so it is difficult to interpret these findings in relation to previous observations . | PMC6331205 | -1 | [] |
The mutations in the cap5A promoter and the cap5D , cap5E and cap5G coding regions [ 55 , 107 ] were also identified in CC8 isolates tested in this study ( Table 3 ) . | PMC6331205 | -1 | [] |
We propose that only one of these mutations ( type 2 , cap5D ) is likely to contribute to the lack of in vitro expression phenotype . | PMC6331205 | -1 | [] |
The cap5D mutation is a frameshift mutation predicted to result in a truncated non functional enzyme due to premature termination of protein synthesis . | PMC6331205 | -1 | [] |
As the cap5A promoter mutation ( mutation type 1 ) was identified in some strains that expressed capsule in vitro , this is unlikely to be the root cause of the acapsular phenotype . | PMC6331205 | -1 | [] |
The cap5E ( mutation type 3 ) and cap5G ( mutation type 4 ) SNPs result in non conservative amino acid substitutions ( Cap5E D75Y and Cap5G F160L ) with the potential to influence the activity of the corresponding enzymes . | PMC6331205 | -1 | [] |
However , Cap5E D75Y maps to a location peripheral to cofactor or substrate binding sites [ 108 ] , and Cap5G F160L falls beyond the boundary of gene fragments capable of genetically complementing the USA300 acapsular phenotype [ 55 ] . | PMC6331205 | -1 | [] |
Additional mutations associated with other CC including CC5 ( cap5E ) ; CC12 ( cap8D and cap8E ) and CC188 ( cap8O ) were also identified in this study . | PMC6331205 | -1 | [] |
In each of the cases tested , there was no correlation between these mutations and the ability of an isolate to express capsule in vivo . | PMC6331205 | -1 | [] |
This was the case , even in the instance of the cap8D frame shift mutation in the two CC12 isolates ( mutation type 6 , Table 3 ) , a mutation that might be expected to ablate expression of capsule functions . | PMC6331205 | -1 | [] |
The third gene where a missense mutation was identified was cap8O in a CC188 isolate . | PMC6331205 | -1 | [] |
This gene has been demonstrated to be essential for the production of D-ManNAcA when it is completely knocked out , however the effects of specific non synonymous substitutions have not been characterized . | PMC6331205 | -1 | [] |
These observations suggest that point / frameshift mutations within the capsular genetic pathway are generally not predictive of the ability of this pathogen to produce capsule during infection . | PMC6331205 | -1 | [] |
The only clear capsule null mutation was identified in a single CC97 isolate within the collection of 516 S. aureus isolates , for which no detectable capsule expression was observed in vitro , suggesting that the acquisition of such mutations is potentially detrimental to S. aureus and thus clonal expansion of such isolates in the clinical setting may be unlikely . | PMC6331205 | -1 | [] |
CC97 is an interesting lineage that is a leading cause of bovine mastitis globally [ 109 , 110 ] and has several major genomic differences to the common S. aureus CC that cause disease in humans . | PMC6331205 | -1 | [] |
The loss of capsule function in the CC97 isolate is consistent with the prevalence of acapsular bovine S. aureus that has evolved through mutations in capsular polysaccharide genes [ 51 , 111 ] . | PMC6331205 | -1 | [] |
Given our data , it seems possible that particular capsular biosynthetic enzymes that are not expressed or are inactive in vitro may be expressed or active in the in vivo environment ; therefore in vitro analyses may not necessarily predict capsule expression in vivo . | PMC6331205 | -1 | [] |
Rozemeijer et al. [ 112 ] reported the detection of RNA transcripts of genes encoding capsular polysaccharide in swab samples from infected patients by qRT-PCR despite the experimental challenges . | PMC6331205 | -1 | [] |
The in vivo mouse experiments presented here have the disadvantage that the number of bacterial isolates that are recoverable is limited , making it difficult to assess the absolute structure of the polysaccharide being detected . | PMC6331205 | -1 | [] |
Despite these limitations , sufficient bacterial RNA was isolated from mouse blood to confirm that individual gene transcripts of the cap operon were being expressed ( Fig 4B ) . | PMC6331205 | -1 | [] |
We acknowledge that we have generated a body of data that can be considered controversial . | PMC6331205 | -1 | [] |
Using specific capsular polysaccharide detection reagents and assays we show that clear differences exist between the expression of capsular polysaccharides by S. aureus disease-causing strains in vivo , compared with expression in the same strains grown in laboratory medium . | PMC6331205 | -1 | [] |
At this point we can only speculate on possible mechanisms . | PMC6331205 | -1 | [] |
Ouyang et al. [ 107 ] were the first to postulate that in vitro expression may be compromised due to a mutation within the cap5A promoter region . | PMC6331205 | -1 | [] |
In this study we have found that this promoter mutation is not limited only to CP5 S. aureus strains that do not express capsule in vitro , it is also present in other strains where capsular polysaccharide expression is detected in vitro . | PMC6331205 | -1 | [] |
A potential explanation for this was outlined by Sau et al. [ 113 ] who demonstrated that while the cap operon was transcribed as a single transcript using the cap5A associated promoter , weaker promoters were also associated with the individual genes . | PMC6331205 | -1 | [] |
Likewise Ouyang et al. [ 107 ] and Gupta et al. [ 114 ] have demonstrated that the principal promoter ( Pcap ) of the cap operon has several different activators and repressors that bind to different promotor sites . | PMC6331205 | -1 | [] |
Genetic and biochemical data may explain how cap5D frameshift mutations associated with USA300 strains can be bypassed in vivo . | PMC6331205 | -1 | [] |
Our transcriptional analyses demonstrates the presence of cap5D transcript during in vivo growth , suggesting that although the translation of the cap5D coding sequence may be disrupted by a frameshift , it is possible that a post-translational bypass mechanism may be responsible for compensating for the cap5D nucleotide insertion under in vivo growth conditions . | PMC6331205 | -1 | [] |
The gene products of capD and capE are similar , with the exception of a membrane bound motif associated with capD , which is thought to anchor the polysaccharide to the cell surface during synthesis [ 115 ] . | PMC6331205 | -1 | [] |
Both proteins encode UDP-GlcNAc C6 dehydratases that are associated with the first steps in D-FucNAc and L-FucNAc biosynthesis , respectively . | PMC6331205 | -1 | [] |
However , Miyafusa et al. [ 108 ] demonstrated that the Cap5E enzyme is capable of producing the stereoisomeric precursors of both D-FucNAc and L-FucNAc in vitro . | PMC6331205 | -1 | [] |
Not only can Cap5E generate the UDP-xylo-sugar ( L-FucNAc precursor ) , but it can also produce the UDP-arabino-sugar ( D-FucNAc precursor ) as byproduct . | PMC6331205 | -1 | [] |
The activity of the enzyme is also likely to be regulated by exogenous metabolites due to the presence of an allosteric site identified in the crystal structure [ 108 ] . | PMC6331205 | -1 | [] |
By generating both D - and L - precursors of fucose , Cap5E has the potential to bypass Cap5D , thus providing a potential mechanism for our observations which could be explored further ( S6 Fig ) . | PMC6331205 | -1 | [] |
In conclusion , our comprehensive study of relevant S. aureus disease isolates demonstrates that although the expression of capsule may not be fully understood in vivo , a much higher proportion of the disease-causing isolates in the US have the potential to express capsule in vivo than previously predicted through analysis of the pathogen cultured in in vitro growth media . | PMC6331205 | -1 | [] |
Studies have identified many genes and pathways involved in capsule production by S. aureus , but the role and mechanisms by which they regulate capsule production during infection remain to be elucidated . | PMC6331205 | -1 | [] |
Our work shows that mutations in the cap operon are identified in a subset of the 16 cap genes and they are associated with specific CC . | PMC6331205 | -1 | [] |
Although all USA300 and some USA500 isolates within the CC8 lineage had the cap5D frameshift mutation , many were able to express capsule in vivo . | PMC6331205 | 157,817 | [
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Phylogenetic analysis of USA300 and USA500 isolates in the context of a wider CC8 population revealed that these two epidemic clones are not directly related [ 89 ] , in contrast to a previous suggestion that USA500 represents a progenitor of the USA300 clone [ 116 ] . | PMC6331205 | 157,817 | [
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In this study , less than 10 % of invasive disease cases were caused by USA300 isolates , where reduced expression of CP in vitro was observed . | PMC6331205 | -1 | [] |
Therefore , surveys of CP prevalence that only take into account the in vitro phenotype or use isolate collections that may not represent the target population for the vaccine , will underestimate the distribution of CP-expressing isolates and thus the potential coverage of CP conjugate containing vaccines . | PMC6331205 | -1 | [] |
The expression of capsule by S. aureus is only one of several virulence mechanisms deployed by this pathogen . | PMC6331205 | -1 | [] |
It is expected that for a vaccine to be effective it will need to also address additional virulence mechanisms such as binding to host structures and an ability to scavenge nutrients [ 33 , 106 , 112 , 117 – 119 ] . | PMC6331205 | -1 | [] |
The appropriate clinical efficacy trials are therefore crucial to demonstrate the effectiveness of a multi-antigen vaccine containing capsular polysaccharide conjugates in preventing S. aureus infections . | PMC6331205 | -1 | [] |
Supporting information . | PMC6331205 | -1 | [] |
S1 Table Genotypic characteristics of S. aureus isolates .. | PMC6331205 | -1 | [] |
( PDF ) Click here for additional data file.S2 Table Frequencies of spa types ( excluding singletons and unknown spa types , n = 93 ) and associated clonal complexes ( CC ) and sequence types ( ST ) .. | PMC6331205 | -1 | [] |
( PDF ) Click here for additional data file.S3 Table Genotypic characteristics of S. aureus strains in the challenge stocks and after recovery from infected mice ( two mice per strain ) in the IFA experiments on in vivo derived bacteria .. | PMC6331205 | -1 | [] |
Presence of the four conserved cap5 operon mutations is indicated in green while the absence of mutations is indicated in red . | PMC6331205 | -1 | [] |
( PDF ) Click here for additional data file.S1 Fig Distribution of clonal complexes ( CC ) of clinical S. aureus isolates collected at different census regions in the US .. | PMC6331205 | -1 | [] |
( TIF ) Click here for additional data file.S2 Fig Distribution of spa types among different healthcare and community-associated infections .. | PMC6331205 | -1 | [] |
( A ) All isolates associated with S. aureus infections in healthcare and community settings . | PMC6331205 | -1 | [] |
( B ) S. aureus isolates associated with the main types of clinical infections in healthcare settings . | PMC6331205 | -1 | [] |
( DOCX ) Click here for additional data file.S3 Fig Distribution of clonal complexes ( CC ) and capsular polysaccharide genotypes ( CP5 / 8) of MRSA and MSSA isolates by year and ward source .. | PMC6331205 | -1 | [] |
( DOCX ) Click here for additional data file.S4 FigApproximately-maximum-likelihood phylogenetic trees of analyzed S. aureus CP5 ( A and C ) and CP8 ( B and D ) isolates annotated with the distribution of cap operon SNPs ( A and B ) and indels in US S. aureus isolates ( C and D ) . | PMC6331205 | -1 | [] |
( A ) cap operon SNPs in CP5 isolates . | PMC6331205 | -1 | [] |
SNPs were identified across the cap operon based on mapping against S. aureus HO 5096 0412 reference . | PMC6331205 | -1 | [] |
Isolate tracks color-coded by CC ( legend below figure ) . | PMC6331205 | -1 | [] |
Color-coding of SNPs ; Green A , Blue G , Black T and Red C. ( B ) cap operon SNPs in CP8 isolates . | PMC6331205 | -1 | [] |
SNPs were identified across the cap operon and plotted against tree of all CP8 USA isolates based on mapping against S. aureus MSSA476 reference . | PMC6331205 | -1 | [] |