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Shigella is a highly prevalent bacterium causing acute diarrhea and dysentery in developing countries. Shigella infections are treated with antibiotics but Shigellae are increasingly resistant to these drugs. Vaccination can be a countermeasure against emerging antibiotic-resistant shigellosis. Because of the structural variability in Shigellae O-antigen polysaccharides (Oag), cross-protective Shigella vaccines cannot be derived from single serotype-specific Oag. We created an attenuated Shigella flexneri 2a strain with one rather than multiple Oag units by disrupting the Oag polymerase gene (Δwzy), which broadened protective immunogenicity by exposing conserved surface proteins. Inactivated Δwzy mutant cells combined with Escherichia coli double mutant LT(R192G/L211A) as adjuvant, induced potent antibody responses to outer membrane protein PSSP-1, and type III secretion system proteins IpaB and IpaC. Intranasal immunization with the vaccine preparation elicited cross-protective immunity against S. flexneri 2a, S. flexneri 3a, S. flexneri 6, and Shigella sonnei in a mouse pneumonia model. Thus, S. flexneri 2a Δwzy represents a promising candidate strain for a universal Shigella vaccine.

Adaptation of bacteria to phage predation poses a major obstacle for phage therapy. Bacteria adopt multiple mechanisms, such as inhibition of phage adsorption and CRISPR/Cas systems, to resist phage infection. Here, a phage-resistant mutant of Pseudomonas aeruginosa strain PA1 under the infection of lytic phage PaP1 was selected for further study. The PaP1-resistant variant, termed PA1RG, showed decreased adsorption to PaP1 and was devoid of long chain O-antigen on its cell envelope. Whole genome sequencing and comparative analysis revealed a single nucleotide mutation in the gene PA1S_08510, which encodes the O-antigen polymerase Wzy that is involved in lipopolysaccharide (LPS) biosynthesis. PA1_Wzy was classified into the O6 serotype based on sequence homology analysis and adopts a transmembrane topology similar to that seem with P. aeruginosa strain PAO1. Complementation of gene wzy in trans enabled the mutant PA1RG to produce the normal LPS pattern with long chain O-antigen and restored the susceptibility of PA1RG to phage PaP1 infection. While wzy mutation did not affect bacterial growth, mutant PA1RG exhibited decreased biofilm production, suggesting a fitness cost of PA1 associated with resistance of phage PaP1 predation. This study uncovered the mechanism responsible for PA1RG resistance to phage PaP1 via wzy mutation and revealed the role of phages in regulating bacterial behavior.

Shigella flexneri utilises the Wzy-dependent pathway for the production of a plethora of complex polysaccharides, including the lipopolysaccharide O-antigen (Oag) component. The inner membrane protein Wzy SF polymerises Oag repeat units, whilst two co-polymerase proteins, Wzz SF and Wzz pHS-2 , together interact with Wzy SF to regulate production of short- (S-Oag) and very long- (VL-Oag) Oag modal lengths, respectively. The 2D arrangement of Wzy SF transmembrane and soluble regions has been previously deciphered, however, attaining information on the 3D structural and conformational arrangement of Wzy SF, or any homologue, has proven difficult. For the first time, the current study detected insights into the in situ Wzy SF arrangement. In vitro assays using thiol-reactive PEG-maleimide were used to probe Wzy SF conformation, which additionally detected novel, unique conformational changes in response to interaction with intrinsic factors, including Wzz SF and Wzz pHS-2 , and extrinsic factors, such as temperature. Site-directed mutagenesis of Wzy SF cysteine residues revealed the presence of a putative intramolecular disulphide bond, between cysteine moieties 13 and 60. Subsequent analyses highlighted both the structural and functional importance of Wzy SF cysteines. Substitution of Wzy SF cysteine residues significantly decreased biosynthesis of the VL-Oag modal length, without disruption to S-Oag production. This phenotype was corroborated in the absence of co-polymerase competition for Wzy SF interaction. These data suggest Wzy SF cysteine substitutions directly impair the interaction between Wzy/Wzz pHS-2, without altering the Wzy/Wzz SF interplay, and in combination with structural data, we propose that the N- and C-termini of Wzy SF are arranged in close proximity, and together may form the unique Wzz pHS-2 interaction site. [Display omitted] • Shigella flexneri utilises the polymerase Wzy SF for LPS O-antigen biosynthesis. • Detection of a novel disulphide bond between Wzy SF moieties C13 and C60. • Identification of novel factor-induced Wzy SF conformational changes in situ. • Optimised approach for membrane protein PEGylation to investigate conformational changes. • Wzy SF NTD and CTD are arranged in close proximity, and form the unique Wzz pHS-2 interaction site. [ABSTRACT FROM AUTHOR]

Shigella strains are human pathogens and their identification is usually based on their O-antigens. The O-antigen gene cluster of Shigella boydii O11 was sequenced. All the expected genes for the synthesis of the O-antigen were identified on the basis of homology and genes for the biosynthesis of dTDP-l-Rhamnose, genes encoding sugar transferases, as well as genes encoding O unit flippase (wzx) and O-antigen polymerase (wzy). The identity of the putative wzy gene was confirmed by showing that a wzy deficient mutant strain of S. boydii O11 produced a semi-rough LPS phenotype. The predicted wzx gene has an opposite transcription direction to that of all of the other genes in the S. boydii O11 O-antigen gene cluster. This unusual feature for the wzx gene has only previously been reported in S. boydii O6. Further comparison revealed an evolutionary relationship between O6 and O11 O-antigen gene clusters. Adjacent-gene PCR showed that Escherichia coli O105 and S. boydii O11, which share the identical O-antigen, also have the same genes and organization for their respective O-antigen gene clusters. Three genes specific for the S. boydii O11 and E. coli O105 gene clusters were identified. [Copyright &y& Elsevier]

Shigella is a highly prevalent bacterium causing acute diarrhea and dysentery in developing countries. Shigella infections are treated with antibiotics but Shigellae are increasingly resistant to these drugs. Vaccination can be a countermeasure against emerging antibiotic-resistant shigellosis. Because of the structural variability in Shigellae O-antigen polysaccharides (Oag), cross-protective Shigella vaccines cannot be derived from single serotype-specific Oag. We created an attenuated Shigella flexneri 2a strain with one rather than multiple Oag units by disrupting the Oag polymerase gene (Δ wzy ), which broadened protective immunogenicity by exposing conserved surface proteins. Inactivated Δ wzy mutant cells combined with Escherichia coli double mutant LT(R192G/L211A) as adjuvant, induced potent antibody responses to outer membrane protein PSSP-1, and type III secretion system proteins IpaB and IpaC. Intranasal immunization with the vaccine preparation elicited cross-protective immunity against S. flexneri 2a, S. flexneri 3a, S. flexneri 6, and Shigella sonnei in a mouse pneumonia model. Thus, S. flexneri 2a Δ wzy represents a promising candidate strain for a universal Shigella vaccine.

Adaptation of bacteria to phage predation poses a major obstacle for phage therapy. Bacteria adopt multiple mechanisms, such as inhibition of phage adsorption and CRISPR/Cas systems, to resist phage infection. Here, a phage-resistant mutant of Pseudomonas aeruginosa strain PA1 under the infection of lytic phage PaP1 was selected for further study. The PaP1-resistant variant, termed PA1RG, showed decreased adsorption to PaP1 and was devoid of long chain O -antigen on its cell envelope. Whole genome sequencing and comparative analysis revealed a single nucleotide mutation in the gene PA1S_08510, which encodes the O -antigen polymerase Wzy that is involved in lipopolysaccharide (LPS) biosynthesis. PA1_Wzy was classified into the O6 serotype based on sequence homology analysis and adopts a transmembrane topology similar to that seem with P. aeruginosa strain PAO1. Complementation of gene wzy in trans enabled the mutant PA1RG to produce the normal LPS pattern with long chain O -antigen and restored the susceptibility of PA1RG to phage PaP1 infection. While wzy mutation did not affect bacterial growth, mutant PA1RG exhibited decreased biofilm production, suggesting a fitness cost of PA1 associated with resistance of phage PaP1 predation. This study uncovered the mechanism responsible for PA1RG resistance to phage PaP1 via wzy mutation and revealed the role of phages in regulating bacterial behavior.