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Emerging B. cereus strains that cause anthrax-like disease have been isolated in Cameroon (CA strain) and Côte d'Ivoire (CI strain). These strains are unusual, because their genomic characterisation shows that they belong to the B. cereus species, although they harbour two plasmids, pBCXO1 and pBCXO2, that are highly similar to the pXO1 and pXO2 plasmids of B. anthracis that encode the toxins and the polyglutamate capsule respectively. The virulence factors implicated in the pathogenicity of these B. cereus bv anthracis strains remain to be characterised. We tested their virulence by cutaneous and intranasal delivery in mice and guinea pigs; they were as virulent as wild-type B. anthracis. Unlike as described for pXO2-cured B. anthracis, the CA strain cured of the pBCXO2 plasmid was still highly virulent, showing the existence of other virulence factors. Indeed, these strains concomitantly expressed a hyaluronic acid (HA) capsule and the B. anthracis polyglutamate (PDGA) capsule. The HA capsule was encoded by the hasACB operon on pBCXO1, and its expression was regulated by the global transcription regulator AtxA, which controls anthrax toxins and PDGA capsule in B. anthracis. Thus, the HA and PDGA capsules and toxins were co-regulated by AtxA. We explored the respective effect of the virulence factors on colonisation and dissemination of CA within its host by constructing bioluminescent mutants. Expression of the HA capsule by itself led to local multiplication and, during intranasal infection, to local dissemination to the adjacent brain tissue. Co-expression of either toxins or PDGA capsule with HA capsule enabled systemic dissemination, thus providing a clear evolutionary advantage. Protection against infection by B. cereus bv anthracis required the same vaccination formulation as that used against B. anthracis. Thus, these strains, at the frontier between B. anthracis and B. cereus, provide insight into how the monomorphic B. anthracis may have emerged.

Background. The capsular polysaccharide (CPS) is an important virulence factor and a vaccine target of the major neonatal pathogen group Streptococcus (GBS). Population studies revealed no strong correlation between CPS type and multilocus sequence typing (MLST) cluster, with the remarkable exception of the worldwide spread of hypervirulent GBS CC17, which were all until recently CPS type III. Methods. A total of 965 GBS strains from invasive infection isolated in France were CPS typed and the presence of the CC17-specific surface protein encoding gene hvgA gene was investigated. Three hvgA-positive GBS strains screened were surprisingly CPS type IV and thus further characterized by MLST typing, pulsed-field gel electrophoresis (PFGE), and whole genome sequencing. Results. MLST and PFGE demonstrated a capsular switching from CPS type III to IV within the highly homogeneous GBS CC17. Sequence analysis revealed that this capsular switch was due to the exchange of a 35.5-kb DNA fragment containing the entire cps operon. Conclusions. This work shows that GBS CCI7 hypervirulent strains have switched one of their main vaccine targets. Thus, continued surveillance of GBS population remains of the utmost importance during clinical trials of conjugate GBS vaccines.

Mutations in the nucleotide-binding oligomerization domain protein 12 (NLRP12) cause recurrent episodes of serosal inflammation. Here we show that NLRP12 efficiently sequesters HSP90 and promotes K48-linked ubiquitination and degradation of NOD2 in response to bacterial muramyl dipeptide (MDP). This interaction is mediated by the linker-region proximal to the nucleotide-binding domain of NLRP12. Consequently, the disease-causing NLRP12 R284X mutation fails to repress MDP-induced NF-κB and subsequent activity of the JAK/STAT signaling pathway. While NLRP12 deficiency renders septic mice highly susceptible towards MDP, a sustained sensing of MDP through NOD2 is observed among monocytes lacking NLRP12. This loss of tolerance in monocytes results in greater colonization resistance towards Citrobacter rodentium . Our data show that this is a consequence of NOD2-dependent accumulation of inflammatory mononuclear cells that correlates with induction of interferon-stimulated genes. Our study unveils a relevant process of tolerance towards the gut microbiota that is exploited by an attaching/effacing enteric pathogen. Mutations in nucleotide-binding oligomerization domain protein 12 (NLRP12) are known to effect inflammatory processes. Here the authors show that NLRP12-mediated proteasomal degradation of NOD2 in monocytes promotes bacterial tolerance and colonisation in a model of enteric infection.

Capsule diversity is a major limiting factor for phage host range in capsulated bacterial hosts. Phage receptor-binding proteins (RBPs) recognize the capsule and initiate infection, making them key players in phage tropism. In this study, we applied an experimental evolution approach to investigate host range adaptation in a diverse 12-phage community interacting with a Klebsiella spp. community containing 39 distinct capsular types. Our findings revealed that generalist phages possessed highly evolvable RBPs, accumulating non-synonymous mutations that modulated their host range. In contrast, specialist phages acquired fewer mutations but remained stable in the community, maintaining their narrow host range. Additionally, recombination between co-infecting closely related phages facilitated rapid host range adaptation through RBP swapping. However, most recombined genes encoded endonucleases or proteins of unknown function, suggesting their potential role in phage survival. This study advances our understanding of phage host range evolution and provides new insights for optimizing phage-based applications.

Hyaluronan is widely used in cosmetics and pharmaceutics. Development of robust and safe cell factories and cultivation approaches to efficiently produce hyaluronan is of many interests. Here, we describe the metabolic engineering of Corynebacterium glutamicum and application of a fermentation strategy to manufacture hyaluronan with different molecular weights. C. glutamicum is engineered by combinatorial overexpression of type I hyaluronan synthase, enzymes of intermediate metabolic pathways and attenuation of extracellular polysaccharide biosynthesis. The engineered strain produces 34.2 g L −1 hyaluronan in fed-batch cultures. We find secreted hyaluronan encapsulates C. glutamicum , changes its cell morphology and inhibits metabolism. Disruption of the encapsulation with leech hyaluronidase restores metabolism and leads to hyper hyaluronan productions of 74.1 g L −1 . Meanwhile, the molecular weight of hyaluronan is also highly tunable. These results demonstrate combinatorial optimization of cell factories and the extracellular environment is efficacious and likely applicable for the production of other biopolymers. Bioproduction of hyaluronan needs increases in yield and greater diversity of the molecular weights. Here, the author increases hyaluronan production and diversifies the molecular weights through engineering the hyaluronan biosynthesis pathway and disruption of Corynebacterium glutamicum encapsulation caused by secreted hyaluronan.

Abstract The ability to produce polysaccharides with diverse biological functions is widespread in bacteria. In lactic acid bacteria (LAB), production of polysaccharides has long been associated with the technological, functional and health-promoting benefits of these microorganisms. In particular, the capsular polysaccharides and exopolysaccharides have been implicated in modulation of the rheological properties of fermented products. For this reason, screening and selection of exocellular polysaccharide-producing LAB has been extensively carried out by academia and industry. To further exploit the ability of LAB to produce polysaccharides, an in-depth understanding of their biochemistry, genetics, biosynthetic pathways, regulation and structure–function relationships is mandatory. Here, we provide a critical overview of the latest advances in the field of glycosciences in LAB. Surprisingly, the understanding of the molecular processes involved in polysaccharide synthesis is lagging behind, and has not accompanied the increasing commercial value and application potential of these polymers. Seizing the natural diversity of polysaccharides for exciting new applications will require a concerted effort encompassing in-depth physiological characterization of LAB at the systems level. Combining high-throughput experimentation with computational approaches, biochemical and structural characterization of the polysaccharides and understanding of the structure–function–application relationships is essential to achieve this ambitious goal. This review describes the recent findings regarding exocellular polysaccharide production in lactic acid bacteria, and provides an overview of their applications in food and future trends in polysaccharide research.

Klebsiella pneumoniae continues to be a substantial public health threat due to its ability to cause health care-associated and community-acquired infections combined with its ability to acquire antibiotic resistance. Novel therapeutics are needed to combat this pathogen, and a greater understanding of its virulence factors is required for the development of new drugs. A key virulence factor for K. pneumoniae is the capsule, and community-acquired hypervirulent strains produce a capsule that causes hypermucoidy. We report here a novel capsule regulator, RmpC, and provide evidence that capsule production and the hypermucoviscosity phenotype are distinct processes. Infection studies showing that this and other capsule regulator mutants have a range of phenotypes indicate that additional virulence factors are in their regulons. These results shed new light on the mechanisms controlling capsule production and introduce targets that may prove useful for the development of novel therapeutics for the treatment of this increasingly problematic pathogen. The polysaccharide capsule is an essential virulence factor for Klebsiella pneumoniae in both community-acquired hypervirulent strains as well as health care-associated classical strains that are posing significant challenges due to multidrug resistance. Capsule production is known to be transcriptionally regulated by a number of proteins, but very little is known about how these proteins collectively control capsule production. RmpA and RcsB are two known regulators of capsule gene expression, and RmpA is required for the hypermucoviscous (HMV) phenotype in hypervirulent K. pneumoniae strains. In this report, we confirmed that these regulators performed their anticipated functions in the ATCC 43816 derivative, KPPR1S: rcsB and rmpA mutants are HMV negative and have reduced capsule gene expression. We also identified a novel transcriptional regulator, RmpC, encoded by a gene near rmpA . The Δ rmpC strain has reduced capsule gene expression but retains the HMV phenotype. We further showed that a regulatory cascade exists in which KvrA and KvrB, the recently characterized MarR-like regulators, and RcsB contribute to capsule regulation through regulation of the rmpA promoter and through additional mechanisms. In a murine pneumonia model, the regulator mutants have a range of colonization defects, suggesting that they regulate virulence factors in addition to capsule. Further testing of the rmpC and rmpA mutants revealed that they have distinct and overlapping functions and provide evidence that HMV is not dependent on overproduction of capsule. This distinction will facilitate a better understanding of HMV and how it contributes to enhanced virulence of hypervirulent strains. IMPORTANCE Klebsiella pneumoniae continues to be a substantial public health threat due to its ability to cause health care-associated and community-acquired infections combined with its ability to acquire antibiotic resistance. Novel therapeutics are needed to combat this pathogen, and a greater understanding of its virulence factors is required for the development of new drugs. A key virulence factor for K. pneumoniae is the capsule, and community-acquired hypervirulent strains produce a capsule that causes hypermucoidy. We report here a novel capsule regulator, RmpC, and provide evidence that capsule production and the hypermucoviscosity phenotype are distinct processes. Infection studies showing that this and other capsule regulator mutants have a range of phenotypes indicate that additional virulence factors are in their regulons. These results shed new light on the mechanisms controlling capsule production and introduce targets that may prove useful for the development of novel therapeutics for the treatment of this increasingly problematic pathogen.

Capsular polysaccharides (CPSs) fortify the cell boundaries of many commensal and pathogenic bacteria 1 . Through the ABC-transporter-dependent biosynthesis pathway, CPSs are synthesized intracellularly on a lipid anchor and secreted across the cell envelope by the KpsMT ABC transporter associated with the KpsE and KpsD subunits 1 , 2 . Here we use structural and functional studies to uncover crucial steps of CPS secretion in Gram-negative bacteria. We show that KpsMT has broad substrate specificity and is sufficient for the translocation of CPSs across the inner bacterial membrane, and we determine the cell surface organization and localization of CPSs using super-resolution fluorescence microscopy. Cryo-electron microscopy analyses of the KpsMT–KpsE complex in six different states reveal a KpsE-encaged ABC transporter, rigid-body conformational rearrangements of KpsMT during ATP hydrolysis and recognition of a glycolipid inside a membrane-exposed electropositive canyon. In vivo CPS secretion assays underscore the functional importance of canyon-lining basic residues. Combined, our analyses suggest a molecular model of CPS secretion by ABC transporters. An ensemble of cryo-electron microscopy structures of the KpsMT ABC transporter in complex with the KpsE co-polymerase and a glycolipid substrate reveal how capsular polysaccharides are recognized and translocated across bacterial cell membranes.

Abstract Porphyromonas gingivalis is a Gram-negative oral anaerobe that is involved in the pathogenesis of periodontitis, an inflammatory disease that destroys the tissues supporting the tooth, eventually leading to tooth loss. Porphyromonas gingivalis has can locally invade periodontal tissues and evade the host defence mechanisms. In doing so, it utilizes a panel of virulence factors that cause deregulation of the innate immune and inflammatory responses. The present review discusses the invasive and evasive strategies of P. gingivalis and the role of its major virulence factors in these, namely lipopolysaccharide, capsule, gingipains and fimbriae. Moreover, the role of P. gingivalis as a ‘keystone’ biofilm species in orchestrating a host response, is highlighted.

Hypervirulent Klebsiella pneumoniae (HvKP) is an emerging bacterial pathogen causing invasive infection in immune-competent humans. The hypervirulence is strongly linked to the overproduction of hypermucoviscous capsule, but the underlying regulatory mechanisms of hypermucoviscosity (HMV) have been elusive, especially at the post-transcriptional level mediated by small noncoding RNAs (sRNAs). Using a recently developed RNA interactome profiling approach iRIL-seq, we interrogate the Hfq-associated sRNA regulatory network and establish an intracellular RNA-RNA interactome in HvKP. Our data reveal numerous interactions between sRNAs and HMV-related mRNAs, and identify a plethora of sRNAs that repress or promote HMV. One of the strongest HMV repressors is ArcZ, which is activated by the catabolite regulator CRP and targets many HMV-related genes including mlaA and fbp . We discover that MlaA and its function in phospholipid transport is crucial for capsule retention and HMV, inactivation of which abolishes Klebsiella virulence in mice. ArcZ overexpression drastically reduces bacterial burden in mice and reduces HMV in multiple hypervirulent and carbapenem-resistant clinical isolates, indicating ArcZ is a potent RNA inhibitor of bacterial pneumonia with therapeutic potential. Our work unravels a novel CRP-ArcZ-MlaA regulatory circuit of HMV and provides mechanistic insights into the posttranscriptional virulence control in a superbug of global concern. By performing a global RNA-RNA interactome analysis in hypervirulent Klebsiella pneumoniae , the authors identify the small RNA ArcZ targets many capsule genes and a key virulence factor MlaA, inhibiting Klebsiella infection and pathogenesis in mice.