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An investigation into the multiple Type VI Secretion Systems of Enterobacter cloacae

(Georgia Institute of Technology, 2021-05) Wilson, Ashley

Enterobacter cloacae is a Gram-negative, opportunistic bacterial pathogen that is commonly acquired by patients in hospitals. The Type VI Secretion System (T6SS) is a harpoon-like apparatus that injects toxins into the cell envelop of neighboring bacteria to defend or compete for resources. It’s commonly found in a range of bacteria including Vibrio cholerae, Pseudomonas aeruginosa, and Escherichia coli. Previous research in this lab has shown that E. cloacae kills in a contact dependent manner and bioinformatic analysis found three vask genes, which encode for a protein in the Type VI apparatus. We created three single mutant strains (Δvask1, Δvask2, Δvask3) as well a double mutant (Δvask1Δvask3). These strains were tested in competition assays with target WT E. coli, with the survival of the target being indicative of E. cloacae killing ability. We show here that Δvask2 had no change in killing ability, Δvask1 and Δvask3 had some reduction in killing ability, and Δvask1Δvask3 had a complete reduction in killing ability. This initial result suggests E. cloacae in vitro killing ability is dependent on two T6SSs.
Emergent collective phenomena in V. Cholerae biofilms, mixed human-autonomous fleets, and nascently multicellular bodies

(Georgia Institute of Technology, 2019-11-07) Yanni, David

Here I focus on the physics of the evolution of multicellularity and division of labor, the cyberphysical risks of hacked internet connected vehicles, and diffusive motion that arises from cell reproduction and lysis (cell death) in biofilms.
Glucose protects E. coli from death by the Vibrio cholerae type VI secretion system

(Georgia Institute of Technology, 2019-05) Nichols, Holly L.

Vibrio cholerae, the causative agent of the intestinal disease cholera, interacts with other bacteria in dense multispecies communities within both host and environmental settings. Using the harpoon-like type VI secretion system (T6SS), V. cholerae delivers toxic effector proteins into neighboring cells, causing cell lysis and death. The T6SS is frequently studied in V. cholerae using a qstR* mutant which constitutively expresses the T6SS. A qstR* V. cholerae strain can effectively kill target species Escherichia coli, Aeromonas veronii., and T6SS-sensistive V. cholerae cells in a standardized lab killing assay, causing a drop in viable cell counts of five orders of magnitude. This study finds that addition of glucose to a standardized killing assay against qstR* V. cholerae restores E. coli survival by three to four orders of magnitude, though the same effect is not found for Aeromonas or T6SS-sensitive V. cholerae. A growth assay revealed that E. coli doubling time does not affect killing by V. cholerae. Additional evidence shows that E. coli does not produce a diffusible molecule that represses the T6SS of V. cholerae. Investigation by fluorescence microscopy revealed that E. coli cells when entirely surrounded by V. cholerae cells survive in the presence but not the absence of glucose, which suggests that glucose causes a relevant physiological change in individual E. coli cells. We propose that further study should focus on the E. coli capsule as a potential mechanism for surviving T6SS attack. This study makes an unprecedented case that attack via the T6SS can be thwarted by sugar metabolism in target cells.
Investigating ice slurry’s perceived mechanical abrasive quality to increase pathogen reduction on poultry during immersion chilling

(Georgia Institute of Technology, 2018-04-26) Richter, Stephanie

The poultry industry is an integral part of Georgia’s economy, accounting for more than half of the state’s agricultural output. In Georgia, more than 20 million pounds of broiler meat are produced daily. Poultry processing entails many phases, and this thesis focuses upon the immersion chilling step. The chilling phase is critical to reducing pathogen presence and ensuring meat product shelf-life. Immersion chilling consumes intense amounts of water and energy resources, and the industry is trying to discover more efficient approaches for processing. This thesis is based upon a multi-year project investigating ice slurry as an alternative chilling medium for the poultry industry. Ice slurry is composed of small characteristic length ice particles and a salt-brine solution that acts as a freezing point depressant. The salt is an important component in maintaining the ice slurry in a homogenous state (i.e., reduce ice agglomeration and media separation). Ice slurry is hypothesized to provide a disruptive scrubbing/abrasive phenomenon resulting in greater pathogen reduction compared to tradition chilled water medium. Pathogen reduction experiments were conducted to determine the Salmonella pathogen reduction capability of chilled water and ice slurry on whole carcasses and wing-parts. Pathogen reduction experiments combined experimental factors of: peracetic acid (PAA) antimicrobial concentration, media salinity, time of immersion chilling, and air agitation levels. Treatment combinations were compared to discover the optimum relationship between factors resulting in the best reduction from STR concentrations pre- and post-chilling. The project also investigated the salt-uptake tendencies of whole carcasses during immersion chilling. Whole carcasses, without the giblets (WOGs) were chilled by either air chill, chilled water, or 4.5% salinity chilled water. Post-chilling, three sample types were collected per each carcass (breast skin, white meat, and dark meat). Results initially addressed salt-uptake concerns when ice slurry medium is used for immersion chilling. Initial findings indicated that salt concentrations increased in the skin, yet did not affect white or dark meat. The skin acts as a barrier that prevents salt penetration into the white and dark meat, furthering the consideration of ice slurry as a poultry chilling medium.
Regulation of the type VI secretion system in environmental isolates of vibrio cholerae

(Georgia Institute of Technology, 2017-12-14) Hoffmann, Tobias

Vibrio cholerae is a human pathogen that causes the severe diarrheal disease cholera, but can also inhabit aquatic environments. The type-VI secretion system (T6SS) is a macromolecular contractile machine that injects neighboring cells with cytotoxic effector proteins. Clinical strains of V. cholerae express the T6SS only when exposed to high cell density and starvation conditions in the presence of chitin, a process regulated by the master regulator QstR. The atypical clinical strain V52 expresses its T6SS constitutively, a trait shared by many V. cholerae strains isolated from the environment. Recently it was discovered that the TfoY regulator controls T6SS expression independent of QstR in V52. In examining strains from environmental sources, I found that one constitutive environmental strain is also under TfoY control. However, I also uncovered that T6SS-mediated constitutive killing in four additional environmental strains was unaffected by a tfoY deletion. Furthermore, I demonstrated that other known regulators (TfoX, QstR, OscR) also played no role in T6SS expression in these strains. For example, the environmental strain BGT69 remains capable of T6SS-mediated killing when these four known T6SS regulators were deleted. These results suggest the presence of a novel regulatory pathway(s) for type-VI secretion in this and other environmental strains of V. cholerae.
Genetics of type VI secretion and natural transformation in Vibrio cholerae

(Georgia Institute of Technology, 2017-01-13) Watve, Samit S.

The facultative waterborne pathogen Vibrio cholerae transitions between its human host and the environment where it colonizes chitinous surfaces in aquatic settings. Growth on chitin coordinates the induction of sets of genes for 1) chitin utilization; 2) a type VI secretion system that allows contact-dependent killing of neighboring bacteria; and 3) DNA uptake by natural transformation, which is a mechanism for horizontal gene transfer. This thesis describes the regulatory network controlling these behaviors in V. cholerae and the consequences of their coordinate regulation. Results from high-throughput RNA sequencing (RNA-seq) show that transcription factor CytR is one of four positive regulators comprising the chitin-induced regulatory network. A combination of genetic and phenotypic assays reveal the four regulators TfoX, HapR, QstR and CytR control each behavior in a distinct manner in a commonly used clinical reference strain of V. cholerae. Whole genome sequencing and bioinformatics analyses of a set of strains isolated from diverse sources reveal novel type VI secretion system components present in environmental, but not clinical isolates. Finally, I show that chitin-induced natural transformation can facilitate horizontal gene transfer of distinct type VI secretion system genes between strains. Horizontally acquired effector-immunity proteins are functional in the new genetic background and can be employed in antibacterial antagonism against parental cells and simultaneously protect against attacks by the donor cells. This thesis sheds light on diverse behavioral adaptations that allow this important human pathogen to spread and persist in the environment.
Natural competence and Type VI secretion in Vibrio cholerae

(Georgia Institute of Technology, 2016-07-22) Bernardy, Eryn

The waterborne bacterium Vibrio cholerae, responsible for epidemics of cholera diarrhea, associates with the human gut and with chitinous surfaces in aquatic reservoirs. Prior studies of two clinical V. cholerae isolates revealed that natural competence for genetic transformation, a horizontal gene transfer mechanism, requires the chitin-induced TfoX regulator, and quorum sensing transcription factor HapR made at high cell density. To further understand this regulation, I helped identify, in a genetic screen, CytR, a new positive regulator required for competence gene expression and natural transformation. Recently, this complex regulatory network in V. cholerae was shown to also control a type VI secretion system (T6SS) that allows contact-dependent killing of other bacteria by injecting toxic proteins. I characterized a diverse set of sequenced V. cholerae isolates, revealing that transformation was rare in all isolates, while constitutive type VI killing was common among environmental but not clinical isolates. These latter results were consistent with a “pathoadaptive” model that tight regulation is beneficial in a host, while constitutive killing is advantageous in the environment. We hypothesized that two sequenced V. cholerae isolates with distinct T6SSs could generate structured populations from initially well-mixed conditions by killing competitors, but not kin. Indeed, when both isolates were rendered T6SS-, a well-mixed population was observed via fluorescence microscopy. In contrast, mutual killing generated clonal patches with each isolate segregating into distinct groups. Structural dynamics were recapitulated with three mathematical models and a cooperation model developed supports that this assortment promotes cooperation among kin. My work in V. cholerae has helped elucidate a complex regulatory network controlling multiple important phenotypes, diversity of these phenotypes among species members, and ecological consequences of antagonistic microbial interactions in the environment.
Developing Tools to Monitor Vibrio cholerae Type VI Secretion During Range Expansion on Solid Surfaces

(Georgia Institute of Technology, 2015-12) Ng, Siu Lung

The waterborne bacterium Vibrio cholerae causes the fatal cholera diarrhea, and thrives in aquatic environments attached to chitinous surfaces with other bacteria. V. cholerae has been used to study natural competence, which promotes DNA uptake through horizontal gene transfer (HGT). A newly described Type VI Secretion System (T6SS) that V. cholerae employ to kill neighboring cells could increase the chance for a competent V. cholerae to take up and utilize the released DNA. The T6SS apparatus, which is similar to a bacteriophage spike, injects toxic effector proteins into prey cells causing lysis that can aid V. cholerae in acquiring DNA from its neighbors. Although previous studies have investigated the regulation and mechanism of T6SS in V. cholerae at a single cell level, the role of T6SS in the dynamics of mixed bacterial populations, such as those found in the environment, remains poorly understood. Fluorescence microscopy was used in this study to determine the effect of T6SS by growing V. cholerae predators and isogenic prey populations on agar surfaces as a model for competitors undergoing spatial expansion. These conditions may mimic conditions encountered on chitinous surfaces in marine settings. In this study, plasmids expressing green and red fluorescent proteins were constructed to visualize the predator and prey populations. Preliminary spatial expansion experiments were ultimately performed using different fluorescent protein alleles that were encoded on the chromosome of competing bacteria. Preliminary results suggest that the presence of a functional T6SS plays an important role in competition. The tools developed for this study are now being used to study interaction between diverse V. cholerae isolates, and as a platform for experimental evolution.
Effects of biofilm production on horizontal gene transfer to Vibrio cholerae

(Georgia Institute of Technology, 2014-05-02) Wilson, Sarah

The waterborne bacterial pathogen Vibrio cholerae utilizes a cell-cell communication system called quorum sensing to coordinate group behavior in both a human host and in aquatic environments. Virulence genes like the cholera toxin, biofilm genes for sticky secreted attachment factors, and competence genes for DNA uptake are all regulated by this population density-dependent system. In a human host, both virulence and biofilm genes are repressed at high cell densities that occur late in infection, presumably to promote transmission upon exhausting the host’s resources. However, in the natural environment, regulation is more complex. Namely, at high cell densities, repression of biofilm production is coordinated with activation of competence genes that can promote horizontal gene transfer (HGT). Based on this model, it was proposed that accumulation of biofilm material on bacterial cells could hinder the uptake of extracellular DNA in aquatic settings. In support of this hypothesis, significant decreases were detected in DNA uptake by V. cholerae strains engineered to overproduce biofilm. However, reductions in DNA uptake were also observed in strains that produced no biofilms. These results suggest that proper timing of biofilm formation plays an important role in the capacity of V. cholerae to engage in HGT, one mechanism thought to allow this pathogen to rapidly evolve in changing environments.
Characterizing a novel direct target of the quorum-sensing controlled small RNAs in V. cholerae

(Georgia Institute of Technology, 2013-12-13) Elsherbini, Joseph Ahmed

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