Capsular Polysaccharide Operon Research Articles

Differential expression of vvhA and CPS operon allele 1 genes in Vibrio vulnificus under biofilm and planktonic conditions

Examination of genes encoding for the virulence factors, hemolysin/cytolysin (vvhA) and capsular polysaccharide (CPS allele 1), during biofilm formation revealed that their expression was influenced by the maturity of the biofilm as well as by temperature. At 24°C, expression of vvhA during biofilm formation was low between 4 and 12h but increased 10-fold by 24h to (5.1 × 104 ± 6.3 × 103mRNA copies/ml) as the biofilm matured. Compared to planktonic cells, expression of vvhA during biofilm formation at 24°C was initially up-regulated at 4h (1.07 ± 0.00-fold) but then was down-regulated almost four-fold during the intermediate and mature stages of biofilm formation. In contrast, vvhA expression at 37°C was up-regulated almost four-fold in the early stages (4 and 6h) of biofilm formation and remained two-fold up-regulated by 24h even as the biofilm was deteriorating. CPS allele 1 expression at 24°C during biofilm formation was up-regulated (1.50 ± 0.18-fold) during the initial attachment phase of the cells but was strongly down-regulated during the intermediate phases at 8 and 10h (74.42 ± 42.16-fold and 453.76 ± 193.32-fold, respectively), indicating that capsular polysaccharide (CPS) is not important to intermediate biofilm architecture. Interestingly, as the biofilm matured by 24h, expression of CPS allele 1 was again up-regulated (1.88 ± 1.07), showing that CPS plays a role in mature biofilm. At 37°C, CPS allele 1 expression was significantly up-regulated (up to 105) during biofilm formation, indicating that the biofilm form of V. vulnificus may be preferred over the planktonic form in the human host.

Comparative Genomic Analysis of Staphylococcus haemolyticus Reveals Key to Hospital Adaptation and Pathogenicity.

Staphylococcus haemolyticus is a skin commensal gaining increased attention as an emerging pathogen of nosocomial infections. However, knowledge about the transition from a commensal to an invasive lifestyle remains sparse and there is a paucity of studies comparing pathogenicity traits between commensal and clinical isolates. In this study, we used a pan-genomic approach to identify factors important for infection and hospital adaptation by exploring the genomic variability of 123 clinical isolates and 46 commensal S. haemolyticus isolates. Phylogenetic reconstruction grouped the 169 isolates into six clades with a distinct distribution of clinical and commensal isolates in the different clades. Phenotypically, multi-drug antibiotic resistance was detected in 108/123 (88%) of the clinical isolates and 5/46 (11%) of the commensal isolates (p < 0.05). In the clinical isolates, we commonly identified a homolog of the serine-rich repeat glycoproteins sraP. Additionally, three novel capsular polysaccharide operons were detected, with a potential role in S. haemolyticus virulence. Clinical S. haemolyticus isolates showed specific signatures associated with successful hospital adaption. Biofilm forming S. haemolyticus isolates that are resistant to oxacillin (mecA) and aminoglycosides (aacA-aphD) are most likely invasive isolates whereas absence of these traits strongly indicates a commensal isolate. We conclude that our data show a clear segregation of isolates of commensal origin, and specific genetic signatures distinguishing the clinical isolates from the commensal isolates. The widespread use of antimicrobial agents has probably promoted the development of successful hospital adapted clones of S. haemolyticus clones through acquisition of mobile genetic elements or beneficial point mutations and rearrangements in surface associated genes.

Biofilm formation in Pseudoalteromonas lipolytica is related to IS5-like insertions in the capsular polysaccharide operon.

Bacterial capsular polysaccharides (CPSs) participate in environmental adaptation in diverse bacteria species. However, the role and regulation of CPS production in marine bacteria have remained largely unexplored. We previously reported that both wrinkled and translucent Pseudoalteromonas lipolytica variants with altered polysaccharide production were generated in pellicle biofilm-associated cells. In this study, we observed that translucent variants were generated at a rate of ∼20% in colony biofilms of P. lipolytica cultured on HSLB agar plates for 12 days. The DNA sequencing results revealed that nearly 90% of these variants had an IS5-like element inserted within the coding or promoter regions of nine genes in the cps operon. In contrast, IS5 insertion into the cps operon was not detected in planktonic cells. Furthermore, we demonstrated that the IS5 insertion event inactivated CPS production, which leads to a translucent colony morphology. The CPS-deficient variants showed an increased ability to form attached biofilms but exhibited reduced resistance to sublethal concentrations of antibiotics. Moreover, deleting the DNA repair gene recA significantly decreased the frequency of occurrence of CPS-deficient variants during biofilm formation. Thus, IS insertion into the cps operon is an important mechanism for the production of genetic variants during biofilm formation of marine bacteria.

Optimization of Molecular Approaches to Genogroup Neisseria meningitidis Carriage Isolates and Implications for Monitoring the Impact of New Serogroup B Vaccines.

The reservoir for Neisseria meningitidis (Nm) is the human oropharynx. Implementation of Nm serogroup C (NmC) glycoconjugate vaccines directly reduced NmC carriage. Prophylactic vaccines are now available to prevent disease caused by the five major Nm disease causing serogroups (ABCWY). Nm serogroup B (NmB) vaccines are composed of antigens that are conserved across Nm serogroups and therefore have the potential to impact all Nm carriage. To assess the effect of these vaccines on carriage, standardized approaches to identify and group Nm are required. Real-time PCR (rt-PCR) capsule grouping assays that were internally controlled to confirm Nm species were developed for eight serogroups associated with carriage (A, B, C, E, W, X, Y and Z). The grouping scheme was validated using diverse bacterial species associated with carriage and then used to evaluate a collection of diverse Nm carriage isolates (n=234). A scheme that also included porA and ctrA probes was able to speciate the isolates, while ctrA also provided insights on the integrity of the polysaccharide loci. Isolates were typed for the Nm vaccine antigen factor H binding protein (fHbp), and were found to represent the known diversity of this antigen. The porA rt-PCR yielded positive results with all 234 of the Nm carriage isolates. Genogrouping assays classified 76.5% (179/234) of these isolates to a group, categorized 53 as nongenogroupable (NGG) and two as mixed results. Thirty seven NGG isolates evidenced a disrupted capsular polysaccharide operon judged by a ctrA negative result. Only 28.6% (67/234) of the isolates were serogrouped by slide agglutination (SASG), highlighting the reduced capability of carriage strains to express capsular polysaccharide. These rt-PCR assays provide a comprehensive means to identify and genogroup N. meningitidis in carriage studies used to guide vaccination strategies and to assess the impact of novel fHbp containing vaccines on meningococcal carriage.

Cyclic AMP and Cyclic AMP-Receptor Protein are Required for Optimal Capsular Polysaccharide Expression

Vibrio vulnificus causes fatal infections in susceptible individuals. Group 1 capsular polysaccharide (CPS) operon is responsible for CPS expression, which plays an essential role in the pathogenesis of this pathogen. Cyclic AMP (cAMP) and cAMP receptor protein (Crp) complex, which responds to glucose availability and functions as a global regulator, has been known to affect CPS production in this pathogen. This study was undertaken to experimentally verify whether cAMP-Crp directly or indirectly affects CPS production. A mutation in cyaA encoding adenylate cyclase, which is required for cAMP biosynthesis, inhibited V. vulnificus growth and changed opaque colonies to translucent colonies, and these changes were recovered by complementing cyaA or by adding exogenous cAMP. A mutation in crp encoding Crp also inhibited V. vulnificus growth and changed opaque colonies to translucent colonies, and these changes were recovered by complementing crp. Moreover, the crp or cyaA mutation decreased the susceptibility of V. vulnificus against NaOCl. The crp mutation reduced the transcription levels of group 1 CPS operon on a per cell basis. Glucose addition in the absence of Crp stimulated V. vulnificus growth, changed translucent colonies to opaque colonies, and increased the transcription levels of group 1 CPS operon. These results indicate that cAMP or Crp is indirectly involved in optimal CPS production by positively affecting metabolism or V. vulnificus growth rather than by directly controlling the expression of group 1 CPS operon.

Comparative genome analysis of Streptococcus infantarius subsp. infantariusCJ18, an African fermented camel milk isolate with adaptations to dairy environment

BackgroundStreptococcus infantarius subsp. infantarius (Sii) belongs to the Streptococcus bovis/Streptococcus equinus complex associated with several human and animal infections. Sii is a predominant bacterium in spontaneously fermented milk products in Africa. The genome sequence of Sii strain CJ18 was compared with that of other Streptococcus species to identify dairy adaptations including genome decay such as in Streptococcus thermophilus, traits for its competitiveness in spontaneous milk fermentation and to assess potential health risks for consumers.ResultsThe genome of Sii CJ18 harbors several unique regions in comparison to Sii ATCC BAA-102T, among others an enlarged exo- and capsular polysaccharide operon; Streptococcus thermophilus-associated genes; a region containing metabolic and hypothetical genes mostly unique to CJ18 and the dairy isolate Streptococcus gallolyticus subsp. macedonicus; and a second oligopeptide transport operon. Dairy adaptations in CJ18 are reflected by a high percentage of pseudogenes (4.9%) representing genome decay which includes the inactivation of the lactose phosphotransferase system (lacIIABC) by multiple transposases integration. The presence of lacS and lacZ genes is the major dairy adaptation affecting lactose metabolism pathways also due to the disruption of lacIIABC.We constructed mutant strains of lacS, lacZ and lacIIABC and analyzed the resulting strains of CJ18 to confirm the redirection of lactose metabolism via LacS and LacZ.Natural competence genes are conserved in both Sii strains, but CJ18 contains a lower number of CRISPR spacers which indicates a reduced defense capability against alien DNA. No classical streptococcal virulence factors were detected in both Sii strains apart from those involved in adhesion which should be considered niche factors. Sii-specific virulence factors are not described. Several Sii-specific regions encoding uncharacterized proteins provide new leads for virulence analyses and investigation of the unclear association of dairy and clinical Sii with human diseases.ConclusionsThe genome of the African dairy isolate Sii CJ18 clearly differs from the human isolate ATCC BAA-102T. CJ18 possesses a high natural competence predisposition likely explaining the enlarged genome. Metabolic adaptations to the dairy environment are evident and especially lactose uptake corresponds to S. thermophilus. Genome decay is not as advanced as in S. thermophilus (10-19%) possibly due to a shorter history in dairy fermentations.

Calcium promotes exopolysaccharide phase variation and biofilm formation of the resulting phase variants in the human pathogen Vibrio vulnificus

Vibrio vulnificus is a Gram-negative bacterium found in estuaries and coastal waters and is associated with human disease caused by ingestion of raw shellfish. Pathogenesis is directly related to the presence of capsular polysaccharide (CPS). Encapsulated virulent strains exhibit an opaque colony phenotype, while unencapsulated attenuated strains appear translucent. A third colony type, rugose, is caused by expression of rugose extracellular polysaccharide (rEPS) and forms robust biofilms. Vibrio vulnificus undergoes phase variation associated with altered levels of CPS and rEPS, and we show here that calcium (Ca²(+) ) significantly increases the rate of CPS and rEPS phase variation in this species. Interestingly, multiple phenotypic responses to increased [Ca²(+) ] were observed among strains, which suggests the existence of underlying cognate genetic or epigenetic differences. Certain translucent isolates contained deletions at the group I CPS operon, inferring increased [Ca²(+) ] upregulates existing phase variation mechanisms. Expanding on a previous observation (Kierek and Watnick, Proc. Natl. Acad. Sci. USA 100: 14357-14362, 2003), increased [Ca²(+) ] also enhanced biofilm formation for all phase variants. Our results show that Ca²(+) promotes both polysaccharide phase variation and biofilm formation of the resulting phase variants, thereby likely serving a dual role in persistence of V. vulnificus in the environment.

Characterization of the type III capsular polysaccharide produced by Burkholderia pseudomallei

Burkholderia pseudomallei has been shown to produce more than one capsular polysaccharide (CPS). Analysis of the B. pseudomallei genome has revealed that the organism contains four CPS operons (I-IV). One of these operons (CPS III) was selected for further study. Comparative sequencing analysis revealed that the genes encoding CPS III are present in B. pseudomallei and Burkholderia thailandensis but not in Burkholderia mallei. In this study, CPS III was not found to contribute to the virulence of B. pseudomallei. Strains containing mutations in CPS III had the same LD(50) value as the wild-type when tested in an animal infection model. Production of CPS III was shown to be induced in water but inhibited in 30% normal human serum using a lux reporter fusion assay. Microarray analysis of capsule gene expression in infected hamsters revealed that the genes encoding CPS III were not significantly expressed in vivo compared with the genes encoding the previously characterized mannoheptose capsule (CPS I), which is an important virulence factor in B. pseudomallei. Glycosyl-composition analysis by combined GC/MS indicated that the CPS III genes are involved in the synthesis of a capsule composed of galactose, glucose, mannose and xylose.

Multiplex PCR assays for simultaneous detection and characterization of Vibrio vulnificus strains

Vibrio vulnificus is a major cause of seafood-related deaths in the United States. Several biomarkers, e.g. the virulence-correlated gene (vcg), 16S rRNA, and the capsular polysaccharide operon (CPS) have been used to differentiate virulent- from nonvirulent-type V. vulnificus strains. In this study, we combined the use of these biomarkers with a species-specific V. vulnificus cytolysin/haemolysin gene (vvhA) to develop two pairs of multiplex PCR assays that simultaneously detect and characterize V. vulnificus strains. The first multiplex PCR pair amplified four genes (vvhA, vcg, 16S rRNA, and CPS), with one for virulent-type and the other one for nonvirulent-type V. vulnificus strains, while the second pair targeted three of those genes excluding CPS. Primer concentration and annealing temperature were optimized for the four multiplex PCR assays. When testing ten V. vulnificus reference strains and 80 field oyster isolates, results from each multiplex PCR matched 100% with known strain characteristics for these target genes. The optimized multiplex PCR assays were capable of simultaneously detecting and characterizing V. vulnificus with high specificity and speed. Multiplex PCR assays designed in this study are valuable tools for microbial ecology and epidemiology studies. They may facilitate better control of V. vulnificus risks in oysters, thereby reducing the number of illnesses and deaths because of V. vulnificus in the long run.

A regulatory RNA required for antitermination of biofilm and capsular polysaccharide operons in Bacillales

Formation of a Bacillus subtilis biofilm community requires an abundant matrix protein, TasA and an exopolysaccharide. The transcriptional regulatory pathways that control synthesis of these structural features are complex and responsive to multiple physiological and population signals. We report herein that an additional layer of co-transcriptional regulation is required for exopolysaccharide (eps) expression. This mechanism is mediated by a novel cis-acting RNA element, coined 'EAR', located between the second and third gene of the eps operon. The presence of the EAR element within the eps operon is required for readthrough of distally located termination signals. We also find that the EAR element promotes readthrough of heterologous termination sites. Based upon these observations, we hypothesize that the EAR element associates with RNA polymerase to promote processive antitermination, a process wherein the transcription elongation complex is altered by accessory factors to become resistant to pausing and termination signals. It is likely that this mechanism is required for eps expression to ensure full synthesis of the unusually long transcript (16 kb). We also identify the EAR element in other species within the order Bacillales, suggesting that a similar mechanism is required for synthesis of biofilm and capsular polysaccharide operons in other microorganisms.

Large scale variation in Enterococcus faecalis illustrated by the genome analysis of strain OG1RF

A comparison of two strains of the hospital pathogen Enterococcus faecalis suggests that mediators of virulence differ between strains and that virulence does not depend on mobile gene elements

Genetic Variation in the Vibrio vulnificus Group 1 Capsular Polysaccharide Operon

Vibrio vulnificus produces human disease associated with raw-oyster consumption or wound infections, but fatalities are limited to persons with chronic underlying illness. Capsular polysaccharide (CPS) is required for virulence, and CPS expression correlates with opaque (Op) colonies that show "phase variation" to avirulent translucent (Tr) phenotypes with reduced CPS. The results discussed here confirmed homology of a V. vulnificus CPS locus to the group 1 CPS operon in Escherichia coli. However, two distinct V. vulnificus genotypes or alleles were associated with the operon, and they diverged at sequences encoding hypothetical proteins and also at unique, intergenic repetitive DNA elements. Phase variation was examined under conditions that promoted high-frequency transition of Op to Tr forms. Recovery of Tr isolates in these experiments showed multiple genotypes, which were designated TR1, TR2, and TR3: CPS operons of TR1 isolates were identical to the Op parent, and cells remained phase variable but expressed reduced CPS. TR2 and TR3 showed deletion mutations in one (wzb) or multiple genes, respectively, and deletion mutants were acapsular and locked in the Tr phase. Complementation in trans restored the Op phenotype in strains with the wzb deletion mutation. Allelic variation in repetitive elements determined the locations, rates, and extents of deletion mutations. Thus, different mechanisms are responsible for reversible phase variation in CPS expression versus genetic deletions in the CPS operon of V. vulnificus. Repetitive-element-mediated deletion mutations were highly conserved within the species and are likely to promote survival in estuarine environments.

CpsK of Streptococcus agalactiae exhibits alpha2,3-sialyltransferase activity in Haemophilus ducreyi.

Streptococcus agalactiae (GBS) is a major cause of serious newborn bacterial infections. Crucial to GBS evasion of host immunity is the production of a capsular polysaccharide (CPS) decorated with sialic acid, which inactivates the alternative complement pathway. The CPS operons of serotypes Ia and III GBS have been described, but the CPS sialyltransferase gene was not identified. We identified cpsK, an open reading frame in the CPS operon of most serotypes, which was homologous to the lipooligosaccharide (LOS) sialyltransferase gene, lst, of Haemophilus ducreyi. To determine if cpsK might encode a sialyltransferase, we complemented a H. ducreyi lst mutant with cpsK. CpsK was expressed in H. ducreyi and LOS was isolated and analysed for sialic acid content by SDS-PAGE and high-performance liquid chromatography (HPLC). Sialo-LOS was seen in the wild-type, cpsK- or lst-complemented mutant strains, but not in the mutant without cpsK. Addition of Neu5Ac to the LOS was confirmed by mass spectroscopy. Lectin binding studies detected terminal Neu5Ac(alpha 2-->3)Gal(beta 1- on LOS produced by the wild-type, cpsK or lst-complemented mutant strain LOS, compared with the mutant alone. Our data characterize the first sialyltransferase gene from a Gram- positive bacterium and provide compelling evidence that its product catalyses the alpha2,3 addition of Neu5Ac to H. ducreyi LOS and therefore the terminal side-chain of GBS CPS. Phylogenetic studies further indicated that lst and cpsK are related but distinct from sialyltransferases of most other bacteria and, along with their similar codon usage bias and G + C content, suggests acquisition by lateral transfer from an ancestral low G + C organism.

Identification of a group 1-like capsular polysaccharide operon for Vibrio vulnificus.

Virulence of Vibrio vulnificus correlates with changes in colony morphology that are indicative of a reversible phase variation for expression of capsular polysaccharide (CPS). Encapsulated variants are virulent with opaque colonies, whereas phase variants with reduced CPS expression are attenuated and are translucent. Using TnphoA mutagenesis, we identified a V. vulnificus CPS locus, which included an upstream ops element, a wza gene (wza(Vv)), and several open reading frames with homology to CPS biosynthetic genes. This genetic organization is characteristic of group 1 CPS operons. The wza gene product is required for transport of CPS to the cell surface in Escherichia coli. Polar transposon mutations in wza(Vv) eliminated expression of downstream biosynthetic genes, confirming operon structure. On the other hand, nonpolar inactivation of wza(Vv) was specific for CPS transport, did not alter CPS biosynthesis, and could be complemented in trans. Southern analysis of CPS phase variants revealed deletions or rearrangements at this locus. A survey of environmental isolates indicated a correlation between deletions in wza(Vv) and loss of virulent phenotype, suggesting a genetic mechanism for CPS phase variation. Full virulence in mice required surface expression of CPS and supported the essential role of capsule in the pathogenesis of V. vulnificus.

Serotype 14 variants of the Spanish penicillin-resistant serotype 9V clone of Streptococcus pneumoniae arose by large recombinational replacements of the cpsA-pbp1a region.

The high prevalence of penicillin resistance among Streptococcus pneumoniae isolates from Uruguay has been associated with the emergence of a penicillin-resistant clone of serotype 14. Isolates of this clone were identical by multilocus sequence typing to members of the Spanish penicillin-resistant serotype 9V clone and possessed indistinguishable forms of the penicillin-binding protein 2b and 2x genes. Their pbp1a genes were also identical, except at the 3' end. The serotype 14 isolates were shown to be a variant of the Spanish serotype 9V clone which arose by a 22.2 kb recombinational replacement that introduced the capsular biosynthetic locus, and part of the neighbouring pbp1a gene, from a serotype 14 isolate. One end of the recombinational replacement was within the first gene of the capsular polysaccharide operon, cpsA, as the sequence of the upstream dexB gene, through most of cpsA, was identical in the penicillin-resistant serotype 9V and 14 isolates, but the sequences differed in the rest of cpsA and in cpsB. The other recombinational junction was at the end of the divergently transcribed pbp1a gene, which is approximately 11.6 kb downstream of the capsular biosynthetic locus. Isolates of this serotype variant were also detected in Spain and Denmark. Penicillin-resistant serotype 14 isolates from Poland were also closely related to the penicillin-resistant serotype 9V clone, but have emerged independently, as one end of the recombinational replacement was upstream of dexB and the other was within pbp1a, but at a different position from that in the serotype 14 variants from Uruguay, Spain and Denmark. Serotype 14 variants of the Spanish serotype 9V clone have therefore arisen on more than one occasion by large recombinational replacements that extend from the start of the cps region into the pbp1a gene.