Dissecting serotype-specific contributions to live oral cholera vaccine efficacy.

The O1 serogroup of Vibrio cholerae causes pandemic cholera and is divided into the Ogawa and Inaba serotypes. The O-antigen is V. cholerae's immunodominant antigen, and the two serotypes, which differ by the presence or absence of a terminally methylated O-antigen, likely influence development of immunity to cholera and oral cholera vaccines (OCVs). However, there is no consensus regarding the relative immunological potency of each serotype, in part because previous studies relied on genetically heterogeneous strains. Here, we engineered matched serotype variants of a live OCV candidate, HaitiV, and used a germfree mouse model to evaluate the immunogenicity and protective efficacy of each vaccine serotype. By combining vibriocidal antibody quantification with single- and mixed-strain infection assays, we found that all three HaitiV variants-InabaV, OgawaV, and HikoV (bivalent Inaba/Ogawa)-were immunogenic and protective. None of the vaccine serotypes were superior across both of these vaccine metrics, suggesting that the impact of O1-serotype variation in OCV design, although detectable, is subtle. However, all three live vaccines significantly outperformed formalin-killed HikoV, supporting the idea that live OCV usage will bolster current cholera control practices. The potency of OCVs was found to be challenge strain-dependent, emphasizing the importance of appropriate strain selection for cholera challenge studies. Our findings and experimental approaches will be valuable for guiding the development of live OCVs and oral vaccines for additional pathogens.

Unsupervised Learning Approach for Comparing Multiple Transposon Insertion Sequencing Studies.

Transposon insertion sequencing (TIS) is a widely used technique for conducting genome-scale forward genetic screens in bacteria. However, few methods enable comparison of TIS data across multiple replicates of a screen or across independent screens, including screens performed in different organisms. Here, we introduce a post hoc analytic framework, comparative TIS (CompTIS), which utilizes unsupervised learning to enable meta-analysis of multiple TIS data sets. CompTIS first implements screen-level principal-component analysis (PCA) and clustering to identify variation between the TIS screens. This initial screen-level analysis facilitates the selection of related screens for additional analyses, reveals the relatedness of complex environments based on growth phenotypes measured by TIS, and provides a useful quality control step. Subsequently, PCA is performed on genes to identify loci whose corresponding mutants lead to concordant/discordant phenotypes across all or in a subset of screens. We used CompTIS to analyze published intestinal colonization TIS data sets from two vibrio species. Gene-level analyses identified both pan-vibrio genes required for intestinal colonization and conserved genes that displayed species-specific requirements. CompTIS is applicable to virtually any combination of TIS screens and can be implemented without regard to either the number of screens or the methods used for upstream data analysis.IMPORTANCE Forward genetic screens are powerful tools for functional genomics. The comparison of similar forward genetic screens performed in different organisms enables the identification of genes with similar or different phenotypes across organisms. Transposon insertion sequencing is a widely used method for conducting genome-scale forward genetic screens in bacteria, yet few bioinformatic approaches have been developed to compare the results of screen replicates and different screens conducted across species or strains. Here, we used principal-component analysis (PCA) and hierarchical clustering, two unsupervised learning approaches, to analyze the relatedness of multiple in vivo screens of pathogenic vibrios. This analytic framework reveals both shared pan-vibrio requirements for intestinal colonization and strain-specific dependencies. Our findings suggest that PCA-based analytics will be a straightforward widely applicable approach for comparing diverse transposon insertion sequencing screens.

The outer membrane is an essential load-bearing element in Gram-negative bacteria.

Gram-negative bacteria possess a complex cell envelope that consists of a plasma membrane, a peptidoglycan cell wall and an outer membrane. The envelope is a selective chemical barrier1 that defines cell shape2 and allows the cell to sustain large mechanical loads such as turgor pressure3. It is widely believed that the covalently cross-linked cell wall underpins the mechanical properties of the envelope4,5. Here we show that the stiffness and strength of Escherichia coli cells are largely due to the outer membrane. Compromising the outer membrane, either chemically or genetically, greatly increased deformation of the cell envelope in response to stretching, bending and indentation forces, and induced increased levels of cell lysis upon mechanical perturbation and during L-form proliferation. Both lipopolysaccharides and proteins contributed to the stiffness of the outer membrane. These findings overturn the prevailing dogma that the cell wall is the dominant mechanical element within Gram-negative bacteria, instead demonstrating that the outer membrane can be stiffer than the cell wall, and that mechanical loads are often balanced between these structures.

A live vaccine rapidly protects against cholera in an infant rabbit model.

Outbreaks of cholera, a rapidly fatal diarrheal disease, often spread explosively. The efficacy of reactive vaccination campaigns-deploying Vibrio cholerae vaccines during epidemics-is partially limited by the time required for vaccine recipients to develop adaptive immunity. We created HaitiV, a live attenuated cholera vaccine candidate, by deleting diarrheagenic factors from a recent clinical isolate of V. cholerae and incorporating safeguards against vaccine reversion. We demonstrate that administration of HaitiV 24 hours before lethal challenge with wild-type V. cholerae reduced intestinal colonization by the wild-type strain, slowed disease progression, and reduced mortality in an infant rabbit model of cholera. HaitiV-mediated protection required viable vaccine, and rapid protection kinetics are not consistent with development of adaptive immunity. These features suggest that HaitiV mediates probiotic-like protection from cholera, a mechanism that is not known to be elicited by traditional vaccines. Mathematical modeling indicates that an intervention that works at the speed of HaitiV-mediated protection could improve the public health impact of reactive vaccination.

Time-Resolved Transposon Insertion Sequencing Reveals Genome-Wide Fitness Dynamics during Infection.

Transposon insertion sequencing (TIS) is a powerful high-throughput genetic technique that is transforming functional genomics in prokaryotes, because it enables genome-wide mapping of the determinants of fitness. However, current approaches for analyzing TIS data assume that selective pressures are constant over time and thus do not yield information regarding changes in the genetic requirements for growth in dynamic environments (e.g., during infection). Here, we describe structured analysis of TIS data collected as a time series, termed pattern analysis of conditional essentiality (PACE). From a temporal series of TIS data, PACE derives a quantitative assessment of each mutant's fitness over the course of an experiment and identifies mutants with related fitness profiles. In so doing, PACE circumvents major limitations of existing methodologies, specifically the need for artificial effect size thresholds and enumeration of bacterial population expansion. We used PACE to analyze TIS samples of Edwardsiella piscicida (a fish pathogen) collected over a 2-week infection period from a natural host (the flatfish turbot). PACE uncovered more genes that affect E. piscicida's fitness in vivo than were detected using a cutoff at a terminal sampling point, and it identified subpopulations of mutants with distinct fitness profiles, one of which informed the design of new live vaccine candidates. Overall, PACE enables efficient mining of time series TIS data and enhances the power and sensitivity of TIS-based analyses.IMPORTANCE Transposon insertion sequencing (TIS) enables genome-wide mapping of the genetic determinants of fitness, typically based on observations at a single sampling point. Here, we move beyond analysis of endpoint TIS data to create a framework for analysis of time series TIS data, termed pattern analysis of conditional essentiality (PACE). We applied PACE to identify genes that contribute to colonization of a natural host by the fish pathogen Edwardsiella piscicida. PACE uncovered more genes that affect E. piscicida's fitness in vivo than were detected using a terminal sampling point, and its clustering of mutants with related fitness profiles informed design of new live vaccine candidates. PACE yields insights into patterns of fitness dynamics and circumvents major limitations of existing methodologies. Finally, the PACE method should be applicable to additional "omic" time series data, including screens based on clustered regularly interspaced short palindromic repeats with Cas9 (CRISPR/Cas9).

Rapid, precise quantification of bacterial cellular dimensions across a genomic-scale knockout library.

BACKGROUND: The determination and regulation of cell morphology are critical components of cell-cycle control, fitness, and development in both single-cell and multicellular organisms. Understanding how environmental factors, chemical perturbations, and genetic differences affect cell morphology requires precise, unbiased, and validated measurements of cell-shape features. RESULTS: Here we introduce two software packages, Morphometrics and BlurLab, that together enable automated, computationally efficient, unbiased identification of cells and morphological features. We applied these tools to bacterial cells because the small size of these cells and the subtlety of certain morphological changes have thus far obscured correlations between bacterial morphology and genotype. We used an online resource of images of the Keio knockout library of nonessential genes in the Gram-negative bacterium Escherichia coli to demonstrate that cell width, width variability, and length significantly correlate with each other and with drug treatments, nutrient changes, and environmental conditions. Further, we combined morphological classification of genetic variants with genetic meta-analysis to reveal novel connections among gene function, fitness, and cell morphology, thus suggesting potential functions for unknown genes and differences in modes of action of antibiotics. CONCLUSIONS: Morphometrics and BlurLab set the stage for future quantitative studies of bacterial cell shape and intracellular localization. The previously unappreciated connections between morphological parameters measured with these software packages and the cellular environment point toward novel mechanistic connections among physiological perturbations, cell fitness, and growth.

Quantitative Imaging of Gut Microbiota Spatial Organization.

Genomic technologies have significantly advanced our understanding of the composition and diversity of host-associated microbial populations. However, their spatial organization and functional interactions relative to the host have been more challenging to study. Here we present a pipeline for the assessment of intestinal microbiota localization within immunofluorescence images of fixed gut cross-sections that includes a flexible software package, BacSpace, for high-throughput quantification of microbial organization. Applying this pipeline to gnotobiotic and human microbiota-colonized mice, we demonstrate that elimination of microbiota-accessible carbohydrates (MACs) from the diet results in thinner mucus in the distal colon, increased proximity of microbes to the epithelium, and heightened expression of the inflammatory marker REG3ß. Measurements of microbe-microbe proximity reveal that a MAC-deficient diet alters monophyletic spatial clustering. Furthermore, we quantify the invasion of Helicobacter pylori into the glands of the mouse stomach relative to host mitotic progenitor cells, illustrating the generalizability of this approach.

De novo morphogenesis in L-forms via geometric control of cell growth.

In virtually all bacteria, the cell wall is crucial for mechanical integrity and for determining cell shape. Escherichia coli's rod-like shape is maintained via the spatiotemporal patterning of cell-wall synthesis by the actin homologue MreB. Here, we transiently inhibited cell-wall synthesis in E. coli to generate cell-wall-deficient, spherical L-forms, and found that they robustly reverted to a rod-like shape within several generations after inhibition cessation. The chemical composition of the cell wall remained essentially unchanged during this process, as indicated by liquid chromatography. Throughout reversion, MreB localized to inwardly curved regions of the cell, and fluorescent cell wall labelling revealed that MreB targets synthesis to those regions. When exposed to the MreB inhibitor A22, reverting cells regrew a cell wall but failed to recover a rod-like shape. Our results suggest that MreB provides the geometric measure that allows E. coli to actively establish and regulate its morphology.

Rod-like bacterial shape is maintained by feedback between cell curvature and cytoskeletal localization.

Cells typically maintain characteristic shapes, but the mechanisms of self-organization for robust morphological maintenance remain unclear in most systems. Precise regulation of rod-like shape in Escherichia coli cells requires the MreB actin-like cytoskeleton, but the mechanism by which MreB maintains rod-like shape is unknown. Here, we use time-lapse and 3D imaging coupled with computational analysis to map the growth, geometry, and cytoskeletal organization of single bacterial cells at subcellular resolution. Our results demonstrate that feedback between cell geometry and MreB localization maintains rod-like cell shape by targeting cell wall growth to regions of negative cell wall curvature. Pulse-chase labeling indicates that growth is heterogeneous and correlates spatially and temporally with MreB localization, whereas MreB inhibition results in more homogeneous growth, including growth in polar regions previously thought to be inert. Biophysical simulations establish that curvature feedback on the localization of cell wall growth is an effective mechanism for cell straightening and suggest that surface deformations caused by cell wall insertion could direct circumferential motion of MreB. Our work shows that MreB orchestrates persistent, heterogeneous growth at the subcellular scale, enabling robust, uniform growth at the cellular scale without requiring global organization.