Capsular Polysaccharide Synthesis Research Articles

A multiplex PCR assay for molecular capsular serotyping of Mannheimia haemolytica serotypes 1, 2, and 6

Mannheimia haemolytica is an important respiratory pathogen of ruminants. Of the 12 capsular serovars identified, 1 and 6 are most frequently associated with disease in cattle, while 2 is largely a commensal. Comparative analysis of 24 M. haemolytica genomes was used to identify unique genes associated with capsular polysaccharide synthesis as amplification targets in a multiplex PCR assay to discriminate between serotype 1, 2, and 6 strains. The specificity of serotype specific gene targets was evaluated against 47 reference strains representing 12 known serovars of M. haemolytica and 101 field isolates identified through antisera agglutination as serotypes 1, 2, or 6. The results suggest this simple and cost-effective serotype specific PCR assay can be used as an alternative to agglutination based techniques to serotype the majority of M. haemolytica collected from bovines, thus averting the need to use animals and invest in expensive sera development for agglutination assays. In addition, the gene targets identified in this study can be used in silico to identify serotype 1, 2, and 6 strains in sequenced M. haemolytica isolates without the need for culture based analysis.

Pneumococcal 23B Molecular Subtype Identified Using Whole Genome Sequencing.

The polysaccharide capsule is a major virulence factor of Streptococcus pneumoniae and the target of all currently licensed pneumococcal vaccines. At present, there are 92 serologically distinct pneumococcal serotypes. Structural and antigenic variation of capsular types is the result of genetic variation within the capsular polysaccharide synthesis (CPS) locus; however, genetic variation may not always result in phenotypic differences which produce novel serotypes. With the introduction of high throughput whole genome sequencing, discovery of novel genotypic variants is not unexpected and this study describes a novel variant of the serotype 23B CPS operon. This novel variant was characterized as a novel genotypic subtype (23B1) with ∼70% homology to the published 23B CPS sequence. High sequence variability was determined in eight cps genes involved in sugar biosynthesis. However, there was no distinction between the classic 23B serotype and 23B1 serologically or in terms of polysaccharide structure. Phylogenetic and eBURST analysis revealed a distinct lineage for 23B1 with multiple clones (UK, Thailand, and USA) that arose at different points during pneumococcal evolution. Analysis of the UK S. pneumoniae isolates (n = 121) revealed an upsurge of 23B1 ST2372 in 2011, after which this previously unseen ST increased to reach 50% proportion of the 23B sequenced isolates from 2013 and remained prevalent within our sequenced isolates from later years. Therefore, although the 23B1 variant appears to have no phenotypic impact and cannot be considered as novel serotype, it appears to have led to a genetic restructuring of the UK serotype 23B population.

Potentially hazardous Streptococcus suis strains latent in asymptomatic pigs in a major swine production area of Thailand.

Carrier pigs have been considered as the major reservoir of Streptococcus suis and couldbe a significant source of human infection. Therefore, we investigated the prevalence and characteristics of latent S. suis in asymptomatic pigs in the pig-farming area of central Thailand, and compared the data to those previously reported in other regions. We collected samples from 340 asymptomatic pigs. S. suis isolates from the samples were confirmed by species-specific PCR (recN PCR). The capsular polysaccharide synthesis gene (cps) types, virulence-associated gene profiles and sequence types (STs) of the isolates were investigated.Results/Key findings. The prevalence of S. suis found in this study was 37 % (125/340 pigs). The most prevalent genotype was mrp-/epf-/sly-. Among the 16 cps-types identified in 135 isolates, cps-type 16 was the most frequent (11 %), whereas 44 % of the isolates were non-typable. In common with the strains causing human sepsis in Thailand, two cps-type 9 isolates and a cps-type 24 isolate from slaughtered pigs belonged to ST16 and ST221, respectively. All the isolated cps-type 2 strains were confirmed as serotype 2 by co-agglutination tests, and these belonged to ST104, the unique ST commonly found in Thai patients; however, in contrast to the endemic areas, the prevalence of serotype 2 strains was relatively low (2 %) and no ST1 isolate was found. Our results showed the population structure differences between S. suis in central Thailand and other regions; however, zoonotic S. suis is certainly latent in asymptomatic pigs in this intensive swine production area.

New insight into chondroitin and heparosan-like capsular polysaccharide synthesis by profiling of the nucleotide sugar precursors.

Escherichia coli K4 and K5 capsular polysaccharides (K4 and K5 CPSs) have been used as starting material for the biotechnological production of chondroitin sulfate (CS) and heparin (HP) respectively. The CPS covers the outer cell wall but in late exponential or stationary growth phase it is released in the surrounding medium. The released CPS concentration was used, so far, as the only marker to connect the strain production ability to the different cultivation conditions employed. Determining also the intracellular UDP-sugar precursor concentration variations, during the bacterial growth, and correlating it with the total CPS production (as sum of the inner and the released ones), could help to better understand the chain biosynthetic mechanism and its bottlenecks. In the present study, for the first time, a new capillary electrophoresis method was set up to simultaneously analyse the UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-glucuronic acid (UDP-GlcA) and the inner CPS portion, extracted at the same time from the bacterial biomasses; separation was performed at 18°C and 18 kV with a borate-based buffer and detection at 200 nm. The E. coli K4 and K5 UDP-sugar pools were profiled, for the first time, at different time points of shake flask growths on a glycerol-containing medium and on the same medium supplemented with the monosaccharide precursors of the CPSs: their concentrations varied from 0.25 to 11 μM·gcdw−1, according to strain, the type of precursor, the growth phase and the cultivation conditions and their availability dramatically influenced the total CPS produced.

Genotyping and investigating capsular polysaccharide synthesis gene loci of non-serotypeable Streptococcus suis isolated from diseased pigs in Canada

Streptococcus suis (S. suis) is an important swine pathogen and an emerging zoonotic agent. Most clinical S. suis strains express capsular polysaccharides (CPS), which can be typed by antisera using the coagglutination test. In this study, 79 S. suis strains recovered from diseased pigs in Canada and which could not be typed using antisera were further characterized by capsular gene typing and sequencing. Four patterns of cps locus were observed: (1) fifteen strains were grouped into previously reported serotypes but presented several mutations in their cps loci, when compared to available data from reference strains; (2) seven strains presented a complete deletion of the cps locus, which would result in an inability to synthesize capsule; (3) forty-seven strains were classified in recently described novel cps loci (NCLs); and (4) ten strains carried novel NCLs not previously described. Different virulence gene profiles (based on the presence of mrp, epf, and/or sly) were observed in these non-serotypeable strains. This study provides further insight in understanding the genetic characteristics of cps loci in non-serotypeable S. suis strains recovered from diseased animals. When using a combination of the previously described 35 serotypes and the complete NCL system, the number of untypeable strains recovered from diseased animals in Canada would be significantly reduced.

Streptococcus suis small RNA rss04 contributes to the induction of meningitis by regulating capsule synthesis and by inducing biofilm formation in a mouse infection model

Streptococcus suis (SS) is an important pathogen for pigs, and it is also considered as a zoonotic agent for humans. Meningitis is one of the most common features of the infection caused by SS, but little is known about the mechanisms of SS meningitis. Recent studies have revealed that small RNAs (sRNAs) have emerged as key regulators of the virulence in several bacteria. In the previous study, we reported that SS sRNA rss04 was up-regulated in pig cerebrospinal fluid and contributes to SS virulence in a zebrafish infection model. Here, we show that rss04 facilitates SS invasion of mouse brain and lung in vivo. Label-free quantitation mass spectrometry analysis revealed that rss04 regulates transcriptional regulator CcpA and several virulence factors including LuxS. Transmission electron microscope and Dot-blot analyses indicated that rss04 represses capsular polysaccharide (CPS) production, which in turn facilitates SS adherence and invasion of mouse brain microvascular endothelial cells bEnd.3 in vitro and activates the mRNA expression of TLR2, CCL2, IL-6 and TNF-α in mouse brain in vivo at 12h post-infection. In addition, rss04 positively regulates SS biofilm formation. Survival analysis of infected mice showed that biofilm state in brain contributes to SS virulence by intracranial subarachnoidal route of infection. Together, our data reveal that SS sRNA rss04 contributes to the induction of meningitis by regulating the CPS synthesis and by inducing biofilm formation, thereby increasing the virulence in a mouse infection model. To our knowledge, rss04 represents the first bacterial sRNA that plays definitive roles in bacterial meningitis.

Patient-Specific Bacteroides Genome Variants in Pouchitis.

ABSTRACTA 2-year longitudinal microbiome study of 22 patients who underwent colectomy with an ileal pouch anal anastomosis detected significant increases in distinct populations of Bacteroides during 9 of 11 patient visits that coincided with inflammation (pouchitis). Oligotyping and metagenomic short-read annotation identified Bacteroides populations that occurred in early samples, bloomed during inflammation, and reappeared after antibiotic treatment. Targeted cultivation of Bacteroides isolates from the same individual at multiple time points and from several patients detected subtle genomic changes, including the identification of rapidly evolving genomic elements that differentiate isogenic strains of Bacteroides fragilis from the mucosa versus lumen. Each patient harbored Bacteroides spp. that are closely related to commonly occurring clinical isolates, including Bacteroides ovatus, B. thetaiotaomicron, B. vulgatus, and B. fragilis, which contained unique loci in different patients for synthesis of capsular polysaccharides. The presence of unique Bacteroides capsular polysaccharide loci within different hosts and between the lumen and mucosa may represent adaptations to stimulate, suppress, and evade host-specific immune responses at different microsites of the ileal pouch.

Effect of kpsM on the virulence of porcine extraintestinal pathogenic Escherichia coli.

In recent years, extraintestinal pathogenic Escherichia coli (ExPEC) has been found to pose a great threat to human and animal health, but its pathogenic mechanism is not fully understood yet. Capsular polysaccharide, an essential virulence factor in these bacteria, can damage the host immune system, and kpsM is a member of the gene cluster responsible for capsular polysaccharide synthesis. In this study, whole sequence alignment of the virulent strain PCN033 and the attenuated strain PCN061 revealed that kpsM exists in PCN033 but not in PCN061. To determine its function and biological characteristics, we deleted kpsM from PCN033 by homologous recombination. The results of adhesion assays, phagocytosis assays and serum bactericidal assays together with the results of colonization assays in mice indicate that the deletion of kpsM decreases the virulence of porcine ExPEC. Our findings about the biological characteristics of kpsM help to elucidate the complex pathogenic mechanism of ExPEC.

CpsJ gene of Streptococcus iniae is involved in capsular polysaccharide synthesis and virulence.

The capsular polysaccharides are an important virulence factor of Streptococcus iniae, protecting the bacterium from destruction and clearance by the immune system. The cpsJ gene encodes a putative UDP-glucose epimerase involved in the capsule synthesis system. To determine the role of the CpsJ protein in the production of the capsule, a ΔcpsJ mutant was generated and analyzed by comparing its growth performances and virulence with those of the wild type (WT) strain. The ΔcpsJ mutant had longer chains, smaller colonies, and a slower growth rate and decreased optical density than the WT, suggesting that the ΔcpsJ mutant produces less capsular polysaccharide. The ΔcpsJ mutant was more able to adhere to and invaded epithelioma papulosum cyprinid cells (EPCs) when its virulence in vitro was compared with that of the WT, but survived less well in the whole blood of channel catfish. When a channel catfish infection model was used to determine the virulence of the ΔcpsJ mutant in vivo, the mutant caused an increase in survival with the mutant (53.33%) versus the WT (26.67%). In summary, mutation of the cpsJ gene influenced both the capsule synthesis and virulence of S. iniae.

Genomic epidemiology and global diversity of the emerging bacterial pathogen Elizabethkingia anophelis.

Elizabethkingia anophelis is an emerging pathogen involved in human infections and outbreaks in distinct world regions. We investigated the phylogenetic relationships and pathogenesis-associated genomic features of two neonatal meningitis isolates isolated 5 years apart from one hospital in Central African Republic and compared them with Elizabethkingia from other regions and sources. Average nucleotide identity firmly confirmed that E. anophelis, E. meningoseptica and E. miricola represent demarcated genomic species. A core genome multilocus sequence typing scheme, broadly applicable to Elizabethkingia species, was developed and made publicly available (http://bigsdb.pasteur.fr/elizabethkingia). Phylogenetic analysis revealed distinct E. anophelis sublineages and demonstrated high genetic relatedness between the African isolates, compatible with persistence of the strain in the hospital environment. CRISPR spacer variation between the African isolates was mirrored by the presence of a large mobile genetic element. The pan-genome of E. anophelis comprised 6,880 gene families, underlining genomic heterogeneity of this species. African isolates carried unique resistance genes acquired by horizontal transfer. We demonstrated the presence of extensive variation of the capsular polysaccharide synthesis gene cluster in E. anophelis. Our results demonstrate the dynamic evolution of this emerging pathogen and the power of genomic approaches for Elizabethkingia identification, population biology and epidemiology.

Bacterial synthesis of polysialic acid lactosides in recombinant Escherichia coli K-12.

Bacterial polysialyltransferases (PSTs) are processive enzymes involved in the synthesis of polysialic capsular polysaccharides. They can also synthesize polysialic acid in vitro from disialylated and trisialylated lactoside acceptors, which are the carbohydrate moieties of GD3 and GT3 gangliosides, respectively. Here, we engineered a non-pathogenic Escherichia coli strain that overexpresses recombinant sialyltransferases and sialic acid synthesis genes and can convert an exogenous lactoside into polysialyl lactosides. Several PSTs were assayed for their ability to synthesize polysialyl lactosides in the recombinant strains. Fed-batch cultures produced α-2,8 polysialic acid or alternate α-2,8-2,9 polysialic acid in quantities reaching several grams per liter. Bacterial culture in the presence of propargyl-β-lactoside as the exogenous acceptor led to the production of conjugatable polysaccharides by means of copper-assisted click chemistry.

A sweet vaccine

Vaccination with a synthetic glycoconjugate, in combination with the administration of an inhibitor that blocks capsular polysaccharide synthesis in bacteria, could offer an alternative route to combat bacterial infections.

Cucumis melo endornavirus: Genome organization, host range and co-divergence with the host

A high molecular weight dsRNA was isolated from a Cucumis melo L. plant (referred to as ‘CL01’) of an unknown cultivar and completely sequenced. Sequence analyses showed that dsRNA is associated with an endornavirus for which a name Cucumis melo endornavirus (CmEV) is proposed. The genome of CmEV-CL01 consists of 15,078 nt, contains a single, 4939 codons-long ORF and terminates with a stretch of 10 cytosine residues. Comparisons of the putative CmEV-encoded polyprotein with available references in protein databases revealed a unique genome organization characterized by the presence of the following domains: viral helicase Superfamily 1 (Hel-1), three glucosyltransferases (doublet of putative capsular polysaccharide synthesis proteins and a putative C_28_Glycosyltransferase), and an RNA-dependent RNA polymerase (RdRp). The presence of three glycome-related domains of different origin makes the genome organization of CmEV unique among endornaviruses. Phylogenetic analyses of viral RdRp domains showed that CmEV belongs to a specific lineage within the family Endornaviridae made exclusively of plant-infecting endornaviruses. An RT-PCR based survey demonstrated high incidence of CmEV among melon germplasm accession (>87% of tested samples). Analyses of partial genome sequences of CmEV isolates from 26 different melon genotypes suggest fine-tuned virus adaptation and co-divergence with the host. Finally, results of the present study revealed that CmEV is present in plants belonging to three different genera in the family Cucurbitaceae. Such diverse host range is unreported for known endornaviruses and suggests a long history of CmEV association with cucurbits predating their speciation.

The degradation of RcsA by ClpYQ(HslUV) protease in Escherichia coli

In Escherichia coli, RcsA, a positive activator for transcription of cps (capsular polysaccharide synthesis) genes, is degraded by the Lon protease. In lon mutant, the accumulation of RcsA leads to overexpression of capsular polysaccharide. In a previous study, overproduction of ClpYQ(HslUV) protease represses the expression of cpsB∷lacZ, but there has been no direct observation demonstrating that ClpYQ degrades RcsA. By means of a MBP-RcsA fusion protein, we showed that RcsA activated cpsB∷lacZ expression and could be rapidly degraded by Lon protease in SG22622 (lon+). Subsequently, the comparative half-life experiments performed in the bacterial strains SG22623 (lon) and AC3112 (lon clpY clpQ) indicated that the RcsA turnover rate in AC3112 was relatively slow and RcsA was stable at 30°C or 41°C. In addition, ClpY could interact with RscA in an in vitro pull-down assay, and the more rapid degradation of RcsA was observed in the presence of ClpYQ protease at 41°C. Thus, we conclude that RcsA is indeed proteolized by ClpYQ protease.

Capsular Polysaccharide (CPS) Release by Serotype 3 Pneumococcal Strains Reduces the Protective Effect of Anti-Type 3 CPS Antibodies.

The efficacy of the serotype 3 (ST3) pneumococcal conjugate vaccine (PCV) remains unclear. While the synthesis of capsular polysaccharide (CPS) of most serotypes is wzy dependent, the strains of two serotypes, 3 and 37, synthesize CPS by the synthase-dependent pathway, resulting in a polysaccharide that is not covalently linked to peptidoglycan and can be released during growth. We hypothesized that the release of CPS during growth reduces anti-type 3 CPS antibody-mediated protection and may explain the lower efficacy of the type 3 component of PCV than that of other PCVs. The in vitro-released CPS concentrations per 10(7) CFU of ST3 and ST37 strains were significantly higher than those for the ST1, ST4, ST6B, and ST14 strains. Following intraperitoneal (i.p.) injection in mice, blood concentrations of CPS were significantly higher for the ST3 than for the ST4/5 strains. The opsonophagocytic killing assay (OPKA) titer of anti-type 3 CPS antibody was significantly reduced by type 3 CPS, culture supernatant, or serum from Streptococcus pneumoniae ST3 strain WU2-infected mice. Mice were injected with capsule-specific antibodies and challenged i.p. with or without the addition of sterile culture supernatant containing type-specific CPS. The addition of 0.2 μl of culture supernatant from WU2 inhibited passive protection, whereas 100-fold-more culture supernatant from S. pneumoniae ST4 strain TIGR4 was required for the inhibition of protection. We conclude that released type 3 CPS interferes with antibody-mediated killing and protection by anti-CPS antibodies. The relative failure of ST3 PCV may be due to CPS release, suggesting that alternative immunization approaches for ST3 may be necessary.

Identification and functional characterization of the putative polysaccharide biosynthesis protein (CapD) of Enterococcus faecium U0317

Most bacterial species produce capsular polysaccharides that contribute to disease pathogenesis through evasion of the host innate immune system and are also involved in inhibiting leukocyte killing. In the present study, we identified a gene in Enterococcus faecium U0317 with homologies to the polysaccharide biosynthesis protein CapD that is made up of 336 amino acids and putatively catalyzes N-linked glycosylation. A capD deletion mutant was constructed and complemented by homologous recombination that was confirmed by PCR and sequencing. The mutant revealed different growth behavior and morphological changes compared to wild-type by scanning electron microscopy, also the capD mutant showed a strong hydrophobicity and that was reversed in the reconstituted mutant. For further characterization and functional analyses, in-vitro cell culture and in-vivo a mouse infection models were used. Antibodies directed against alpha lipotechoic acid (αLTA) and the peptidyl-prolyl cis-trans isomerase (αPpiC), effectively mediated the opsonophagocytic killing in the capD knock-out mutant, while this activity was not observed in the wild-type and reconstituted mutant. By comparison more than 2-fold decrease was seen in mutant colonization and adherence to both T24 and Caco2 cells. However, a significant higher bacterial colonization was observed in capD mutant during bacteremia in the animal model, while virulence in a mouse UTI (urinary tract infection) model, there were no obvious differences. Further studies are needed to elucidate the function of capsular polysaccharide synthesis gene clusters and its involvement in the disease pathogenesis with the aim to develop targeted therapies to treat multidrug-resistant E. faecium infections.

Genetic analysis of capsular polysaccharide synthesis gene clusters in 79 capsular types of Klebsiella spp.

A total of 79 capsular types have been reported in Klebsiella spp., whereas capsular polysaccharide synthesis (cps) regions were available in only 22 types. Due to the limitations of serotyping, complete repertoire of cps will be helpful for capsular genotyping. We therefore resolved the rest 57 cps and conducted comparative analysis. Clustering results of 1,515 predicted proteins from cps loci categorized proteins which share similarity into homology groups (HGs) revealing that 77 Wzy polymerases were classified into 56 HGs, which indicate the high specificity of wzy between different types. Accordingly, wzy-based capsular genotyping could differentiate capsule types except for those lacking wzy (K29 and K50), those sharing identical wzy (K22 vs. K37); and should be carefully applied in those exhibited high similarity (K12 vs. K41, K2 vs. K13, K74 vs. K80, K79 vs. KN1 and K30 vs. K69). Comparison of CPS structures in several capsular types that shared similarity in their gene contents implies possible functions of glycosyltransferases. Therefore, our results provide complete set of cps in various types of Klebsiella spp., which enable the understandings of relationship between genes and CPS structures and are useful for identification of documented or new capsular types.

Discovery and characterization of natural tropolones as inhibitors of the antibacterial target CapF from Staphylococcus aureus.

The rapid spread of antibiotic-resistance among pathogenic bacteria poses a serious risk for public health. The search for novel therapeutic strategies and antimicrobial compounds is needed to ameliorate this menace. The bifunctional metalloenzyme CapF is an antibacterial target produced by certain pathogenic bacteria essential in the biosynthetic route of capsular polysaccharide, a mucous layer on the surface of bacterium that facilitates immune evasion and infection. We report the first inhibitor of CapF from Staphylococcus aureus, which was identified by employing fragment-based methodologies. The hit compound 3-isopropenyl-tropolone inhibits the first reaction catalyzed by CapF, disrupting the synthesis of a key precursor of capsular polysaccharide. Isothermal titration calorimetry demonstrates that 3-isopropenyl-tropolone binds tightly (KD = 27 ± 7 μM) to the cupin domain of CapF. In addition, the crystal structure of the enzyme-inhibitor complex shows that the compound engages the essential Zn2+ ion necessary for the first reaction catalyzed by the enzyme, explaining its inhibitory effect. Moreover, the tropolone compound alters the coordination sphere of the metal, leading to the overall destabilization of the enzyme. We propose 3-isopropenyl-tropolone as a precursor to develop stronger inhibitors for this family of enzymes to impair the synthesis of capsular polysaccharide in Staphylococcus aureus.

Suppression of capsule expression in Δlon strains of Escherichia coli by two novel rpoB mutations in concert with HNS: possible role for DNA bending at rcsA promoter.

Analyses of mutations in genes coding for subunits of RNA polymerase always throw more light on the intricate events that regulate the expression of gene(s). Lon protease of Escherichia coli is implicated in the turnover of RcsA (positive regulator of genes involved in capsular polysaccharide synthesis) and SulA (cell division inhibitor induced upon DNA damage). Failure to degrade RcsA and SulA makes lon mutant cells to overproduce capsular polysaccharides and to become sensitive to DNA damaging agents. Earlier reports on suppressors for these characteristic lon phenotypes related the role of cochaperon DnaJ and tmRNA. Here, we report the isolation and characterization of two novel mutations in rpoB gene capable of modulating the expression of cps genes in Δlon strains of E. coli in concert with HNS. clpA, clpB, clpY, and clpQ mutations do not affect this capsule expression suppressor (Ces) phenotype. These mutant RNA polymerases affect rcsA transcription, but per se are not defective either at rcsA or at cps promoters. The results combined with bioinformatics analyses indicate that the weaker interaction between the enzyme and DNA::RNA hybrid during transcription might play a vital role in the lower level expression of rcsA. These results might have relevance to pathogenesis in related bacteria.

Phenotypic heterogeneity and temporal expression of the capsular polysaccharide in Staphylococcus aureus.

Bacteria respond to ever-changing environments through several adaptive strategies. This includes mechanisms leading to a high degree of phenotypic variability within a genetically homogeneous population. In Staphylococcus aureus, the capsular polysaccharide (CP) protects against phagocytosis, but also impedes adherence to endothelial cells and/or matrix proteins. We analysed the regulation of core biosynthesis genes (capA-P) necessary for CP synthesis using single-cell assays (immunofluorescence and promoter-activity). In persistent human carriers, we found a distinct subpopulation of nasal S. aureus to be CP positive. In vitro, cap expression is also heterogeneous and strongly growth-phase dependent. We asked whether this peculiar expression pattern (earlyOff/lateHeterogen) is orchestrated by the quorum system Agr. We show that the Agr-driven effector molecule RNAIII promotes cap expression largely via inactivation of the repressor Rot. High NaCl, deletion of CodY or Sae also resulted in higher cap expression but did not change the earlyOFF/lateHeterogen expression pattern. Activity of the quorum system itself is largely homogenous and does not account for the observed heterogeneity of cap expression or the strictly growth phase dependent expression. Our findings are in contrast to the prevailing view that quorum sensing is the main driving force for virulence gene expression when bacterial cell densities increase.