Mutational Analysis of the Shigella flexneri O-Antigen Polymerase Wzy: Identification of Wzz-Dependent Wzy Mutants

The O-antigen (Oag) component of lipopolysaccharide (LPS) is a major virulence determinant ofShigella flexneriand is synthesized by the O-antigen polymerase, WzySf. Oag chain length is regulated by chromosomally encoded WzzSfand pHS-2 plasmid-encoded WzzpHS2. To identify functionally important amino acid residues in WzySf, random mutagenesis was performed on thewzySfgene in a pWaldo-TEV-GFP plasmid, followed by screening with colicin E2. Analysis of the LPS conferred by mutated WzySfproteins in thewzySf-deficient (Δwzy) strain identified 4 different mutant classes, with mutations found in periplasmic loop 1 (PL1), PL2, PL3, and PL6, transmembrane region 2 (TM2), TM4, TM5, TM7, TM8, and TM9, and cytoplasmic loop 1 (CL1) and CL5. The association of WzySfand WzzSfwas investigated by transforming these mutatedwzySfplasmids into awzySf- andwzzSf-deficient (Δwzy Δwzz) strain. Comparison of the LPS profiles in the Δwzyand Δwzy Δwzzbackgrounds identified WzySfmutants whose polymerization activities were WzzSfdependent. Colicin E2 and bacteriophage Sf6c sensitivities were consistent with the LPS profiles. Analysis of the expression levels of the WzySf-GFP mutants in the Δwzyand Δwzy Δwzzbackgrounds identified a role for WzzSfin WzySfstability. Hence, in addition to its role in regulating Oag modal chain length, WzzSfalso affects WzySfactivity and stability.

Relationship between O-antigen chain length and resistance to colicin E2 in Shigella flexneri

The Shigella flexneri polysaccharide co-polymerase class 1a (PCP1a) protein, WzzBSF, regulates LPS O-antigen (Oag) chain length to confer short (S)-type Oag chains of ~10–17 Oag repeat units (RUs). The S-type Oag chains affect Shigella flexneri virulence as they influence IcsA-mediated actin-based motility. However, they do not confer resistance to complement; this is conferred by the very-long (VL)-type Oag chains determined by WzzBpHS2. Colicins are bacterial proteins produced by some Escherichia coli strains to kill related strains. While the presence of Oag chains has been shown to shield outer-membrane proteins from colicins, the impact of Oag chain length against colicins is unknown. In this study, initial testing indicated that a Shigella flexneri Y wzz : : kanr mutant was more sensitive to colicin E2 compared with the WT strain. Plasmids encoding Wzz mutant and WT PCP1a proteins conferring different Oag modal chain lengths were then expressed in the mutant background, and tested against purified colicin E2. Analysis of swab and spot sensitivity assays showed that strains expressing either S-type or long (L)-type Oag chains (16–28 Oag RUs) conferred greater resistance to colicin E2 compared with strains having very-short-type (2–8 Oag RUs), intermediate-short-type (8–14 Oag RUs) or VL-type (>80 Oag RUs) Oag chains. These results suggest a novel role for LPS Oag chain length control that may have evolved due to selection pressure from colicins in the environment.

Colicin E2 Expression in Lactobacillus brevis DT24, A Vaginal Probiotic Isolate, against Uropathogenic Escherichia coli

Novel therapeutic approaches are needed to combat the urinary tract infection in women. During menstruation elevated protein concentration and increase in oxygen and carbon dioxide concentrations with decrease in vaginal Lactobacilli all together contribute to urinary tract infections. Lactobacillus species are a predominant member of the vaginal microflora and are critical in the prevention of a number of urogenital diseases. In order to increase antimicrobial potential of vaginal Lactobacilli, bacteriocin colicin E2 which has specific activity against uropathogenic Escherichia coli has been overexpressed in vaginal probiotic Lactobacillus brevis DT24. Recombinant Lactobacillus brevis DT24 expressing colicin E2 showed much higher inhibitory activity against uropathogenic Escherichia coli than wild type L. brevis DT24 in vitro. Efficacy of probiotic Lactobacillus brevis DT24 expressing colicin E2 protein is required for further in vivo evaluation.

YieJ (CbrC) Mediates CreBC-Dependent Colicin E2 Tolerance in Escherichia coli

ABSTRACT Colicin E2-tolerant (known as Cet2) Escherichia coli K-12 mutants overproduce an inner membrane protein, CreD, which is believed to cause the Cet2 phenotype. Here, we show that overproduction of CreD in a Cet2 strain results from hyperactivation of the CreBC two-component regulator, but CreD overproduction is not responsible for the Cet2 phenotype. Through microarray analysis and gene knockout and overexpression studies, we show that overexpression of another CreBC-regulated gene, yieJ (also known as cbrC), causes the Cet2 phenotype.