Suppressor Mutations in Type II Secretion Mutants of Vibrio cholerae: Inactivation of the VesC Protease.

The type II secretion system (T2SS) is a conserved transport pathway responsible for the secretion of a range of virulence factors by many pathogens, including Vibrio cholerae Disruption of the T2SS genes in V. cholerae results in loss of secretion, changes in cell envelope function, and growth defects. While T2SS mutants are viable, high-throughput genomic analyses have listed these genes among essential genes. To investigate whether secondary mutations arise as a consequence of T2SS inactivation, we sequenced the genomes of six V. cholerae T2SS mutants with deletions or insertions in either the epsG, epsL, or epsM genes and identified secondary mutations in all mutants. Two of the six T2SS mutants contain distinct mutations in the gene encoding the T2SS-secreted protease VesC. Other mutations were found in genes coding for V. cholerae cell envelope proteins. Subsequent sequence analysis of the vesC gene in 92 additional T2SS mutant isolates identified another 19 unique mutations including insertions or deletions, sequence duplications, and single-nucleotide changes resulting in amino acid substitutions in the VesC protein. Analysis of VesC variants and the X-ray crystallographic structure of wild-type VesC suggested that all mutations lead to loss of VesC production and/or function. One possible mechanism by which V. cholerae T2SS mutagenesis can be tolerated is through selection of vesC-inactivating mutations, which may, in part, suppress cell envelope damage, establishing permissive conditions for the disruption of the T2SS. Other mutations may have been acquired in genes encoding essential cell envelope proteins to prevent proteolysis by VesC.IMPORTANCE Genome-wide transposon mutagenesis has identified the genes encoding the T2SS in Vibrio cholerae as essential for viability, but the reason for this is unclear. Mutants with deletions or insertions in these genes can be isolated, suggesting that they have acquired secondary mutations that suppress their growth defect. Through whole-genome sequencing and phenotypic analysis of T2SS mutants, we show that one means by which the growth defect can be suppressed is through mutations in the gene encoding the T2SS substrate VesC. VesC homologues are present in other Vibrio species and close relatives, and this may be why inactivation of the T2SS in species such as Vibrio vulnificus, Vibrio sp. strain 60, and Aeromonas hydrophila also results in a pleiotropic effect on their outer membrane assembly and integrity.

Prevalence of Infection-Competent Serogroup 6 Legionella pneumophila within Premise Plumbing in Southeast Michigan.

Coinciding with major changes to its municipal water system, Flint, MI, endured Legionnaires' disease outbreaks in 2014 and 2015. By sampling premise plumbing in Flint in the fall of 2016, we found that 12% of homes harbored legionellae, a frequency similar to that in residences in neighboring areas. To evaluate the genetic diversity of Legionella pneumophila in Southeast Michigan, we determined the sequence type (ST) and serogroup (SG) of the 18 residential isolates from Flint and Detroit, MI, and the 33 clinical isolates submitted by hospitals in three area counties in 2013 to 2016. Common to one environmental and four clinical samples were strains of L. pneumophila SG1 and ST1, the most prevalent ST worldwide. Among the Flint premise plumbing isolates, 14 of 16 strains were of ST367 and ST461, two closely related SG6 strain types isolated previously from patients and corresponding environmental samples. Each of the representative SG1 clinical strains and SG6 environmental isolates from Southeast Michigan infected and survived within macrophage cultures at least as well as a virulent laboratory strain, as judged by microscopy and by enumerating CFU. Likewise, 72 h after infection, the yield of viable-cell counts increased >100-fold for each of the representative SG1 clinical isolates, Flint premise plumbing SG6 ST367 and -461 isolates, and two Detroit residential isolates. We verified by immunostaining that SG1-specific antibody does not cross-react with the SG6 L. pneumophila environmental strains. Because the widely used urinary antigen diagnostic test does not readily detect non-SG1 L. pneumophila, Legionnaires' disease caused by SG6 L. pneumophila is likely underreported worldwide.IMPORTANCEL. pneumophila is the leading cause of disease outbreaks associated with drinking water in the United States. Compared to what is known of the established risks of colonization within hospitals and hotels, relatively little is known about residential exposure to L. pneumophila One year after two outbreaks of Legionnaires' disease in Genesee County, MI, that coincided with damage to the Flint municipal water system, our multidisciplinary team launched an environmental surveillance and laboratory research campaign aimed at informing risk management strategies to provide safe public water supplies. The most prevalent L. pneumophila strains isolated from residential plumbing were closely related strains of SG6. In laboratory tests of virulence, the SG6 environmental isolates resembled SG1 clinical strains, yet they are not readily detected by the common diagnostic urinary antigen test, which is specific for SG1. Therefore, our study complements the existing epidemiological literature indicating that Legionnaires' disease due to non-SG1 strains is underreported around the globe.

Variation in germination of Clostridium difficile clinical isolates correlates to disease severity.

Over the past two decades, Clostridium difficile infections have been increasing in both number and severity throughout the world. As with other spore forming bacteria, germination is a vital step in the life cycle of this pathogen. Studies have examined differences in sporulation and toxin production among a number of C. difficile clinical isolates; however, few have examined differences in germination and the relationship between this phenotype and disease severity. Here, over 100 C. difficile isolates from the University of Michigan Health System were examined for overall germination in response to various combinations of known germinants (taurocholate) and co-germinants (glycine and histidine). Significant variation was observed among isolates under all conditions tested. Isolates representing ribotype 014-020, which was the most frequently isolated ribotype at our hospital, exhibited increased germination in the presence of taurocholate and glycine when compared to isolates representing other ribotypes. Interestingly, isolates that caused severe disease exhibited significantly lower germination in response to minimal germination conditions (taurocholate only), indicating increased control over germination in these isolates. These data provide a broad picture of C. difficile isolate germination and indicate a role for precise control of germination in disease severity.