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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.

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.

Acinetobacter baumannii is an opportunistic pathogen of increasing importance due to its propensity for intractable multidrug-resistant infections in hospitals. All clinical isolates examined contain a conserved gene cluster, the K locus, which determines the production of complex polysaccharides, including an exopolysaccharide capsule known to protect against killing by host serum and to increase virulence in animal models of infection. Whether the polysaccharides determined by the K locus contribute to intrinsic defenses against antibiotics is unknown. We demonstrate here that mutants deficient in the exopolysaccharide capsule have lowered intrinsic resistance to peptide antibiotics, while a mutation affecting sugar precursors involved in both capsule and lipopolysaccharide synthesis sensitizes the bacterium to multiple antibiotic classes. We observed that, when grown in the presence of certain antibiotics below their MIC, including the translation inhibitors chloramphenicol and erythromycin, A. baumannii increases production of the K locus exopolysaccharide. Hyperproduction of capsular exopolysaccharide is reversible and non-mutational, and occurs concomitantly with increased resistance to the inducing antibiotic that is independent of the presence of the K locus. Strikingly, antibiotic-enhanced capsular exopolysaccharide production confers increased resistance to killing by host complement and increases virulence in a mouse model of systemic infection. Finally, we show that augmented capsule production upon antibiotic exposure is facilitated by transcriptional increases in K locus gene expression that are dependent on a two-component regulatory system, bfmRS. These studies reveal that the synthesis of capsule, a major pathogenicity determinant, is regulated in response to antibiotic stress. Our data are consistent with a model in which gene expression changes triggered by ineffectual antibiotic treatment cause A. baumannii to transition between states of low and high virulence potential, which may contribute to the opportunistic nature of the pathogen.

Among the most urgent public health threats is the worldwide emergence of carbapenem-resistant Enterobacteriaceae 1 – 4 , which are resistant to the antibiotic class of ‘last resort’. In the United States and Europe, carbapenem-resistant strains of the Klebsiella pneumoniae ST258 (ref. 5 ) sequence type are dominant, endemic 6 – 8 and associated with high mortality 6 , 9 , 10 . We report the global evolution of pathogenicity in carbapenem-resistant K. pneumoniae , resulting in the repeated convergence of virulence and carbapenem resistance in the United States and Europe, dating back to as early as 2009. We demonstrate that K. pneumoniae can enhance its pathogenicity by adopting two opposing infection programs through easily acquired gain- and loss-of-function mutations. Single-nucleotide polymorphisms in the capsule biosynthesis gene wzc lead to hypercapsule production, which confers phagocytosis resistance, enhanced dissemination and increased mortality in animal models. In contrast, mutations disrupting capsule biosynthesis genes impair capsule production, which enhances epithelial cell invasion, in vitro biofilm formation and persistence in urinary tract infections. These two types of capsule mutants have emerged repeatedly and independently in Europe and the United States, with hypercapsule mutants associated with bloodstream infections and capsule-deficient mutants associated with urinary tract infections. In the latter case, drug-tolerant K. pneumoniae can persist to yield potentially untreatable, persistent infection. Mutations in Klebsiella pneumoniae that lead to gain or loss of capsule production affect pathogenicity and associate with bloodstream or urinary tract infections, respectively.

The genomes of human gut bacteria in the genus Bacteroides include numerous operons for biosynthesis of diverse capsular polysaccharides (CPSs). The first two genes of each CPS operon encode a locus-specific paralog of transcription elongation factor NusG (called UpxY), which enhances transcript elongation, and a UpxZ protein that inhibits noncognate UpxYs. This process, together with promoter inversions, ensures that a single CPS operon is transcribed in most cells. Here, we use in-vivo nascent-RNA sequencing and promoter-less in-vitro transcription (PIVoT) to show that UpxY recognizes a paused RNA polymerase via sequences in both the exposed non-template DNA and the upstream duplex DNA. UpxY association is aided by ‘pause-then-escape’ nascent RNA hairpins. UpxZ binds non-cognate UpxYs to directly inhibit UpxY association. This UpxY-UpxZ hierarchical regulatory program allows Bacteroides to generate subpopulations of cells producing diverse CPSs for optimal fitness. Bacteroides strains have multiple operons for biosynthesis of diverse capsular polysaccharides, but most cells express only one operon at a time due to tight regulation of transcription elongation by locus-specific UpxY and UpxZ proteins. Here, Saba et al. provide insight into the mechanisms by which UpxY distinguishes among cognate operons and how UpxZ inhibits only noncognate UpxY proteins.

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.

Diversity of the polysaccharide capsule in Streptococcus pneumoniae—main surface antigen and the target of the currently used pneumococcal vaccines—constitutes a major obstacle in eliminating pneumococcal disease. Such diversity is genetically encoded by almost 100 variants of the capsule biosynthesis locus, cps. However, the evolutionary dynamics of the capsule remains not fully understood. Here, using genetic data from 4,519 bacterial isolates, we found cps to be an evolutionary hotspot with elevated substitution and recombination rates. These rates were a consequence of relaxed purifying selection and positive, diversifying selection acting at this locus, supporting the hypothesis that the capsule has an increased potential to generate novel diversity compared with the rest of the genome. Diversifying selection was particularly evident in the region of wzd/wze genes, which are known to regulate capsule expression and hence the bacterium’s ability to cause disease. Using a novel, capsule-centered approach, we analyzed the evolutionary history of 12 major serogroups. Such analysis revealed their complex diversification scenarios, which were principally driven by recombination with other serogroups and other streptococci. Patterns of recombinational exchanges between serogroups could not be explained by serotype frequency alone, thus pointing to nonrandom associations between co-colonizing serotypes. Finally, we discovered a previously unobserved mosaic serotype 39X, which was confirmed to carry a viable and structurally novel capsule. Adding to previous discoveries of other mosaic capsules in densely sampled collections, these results emphasize the strong adaptive potential of the bacterium by its ability to generate novel antigenic diversity by recombination.

The capsule is a major virulence factor of Streptococcus pneumoniae ( Spn ), providing a physical shield and exhibiting extensive diversity across at least 100 serotypes. Although natural transformation of Spn has predominantly been characterized in unencapsulated laboratory strains, clinical encapsulated isolates also exhibit transformability and demonstrate varied recombination rates during host carriage. We utilized otherwise genetically identical capsule-switch strains to isolate the effect of capsule on transformation. We demonstrate serotype- and quantity-dependent inhibition of transformation by the capsule, mediated through hindrance with the transformation pilus assembly and function. This study challenges the paradigm that unencapsulated laboratory strains fully recapitulate natural transformation dynamics. By redefining the capsule as a multifunctional modulator of Spn biology, balancing virulence and adaptability, our findings advance our understanding of pneumococcal evolution.