Date of Completion

2024

Document Type

Honors College Thesis

Department

Microbiology and Molecular Genetics

Thesis Type

Honors College

First Advisor

Leigh Knodler, Ph.D

Second Advisor

Princess Rodriguez, Ph.D

Third Advisor

Jean Celli, Ph.D

Keywords

Providencia, Bioinformatics, Bacterial Killing Assay, Mammalian Infection

Abstract

The Providencia genus is a member of the Enterobacteriaceae family of bacteria and is a set of emerging, opportunistic pathogens that are known to cause urinary tract infections and gastroenteritis (Shah et al., 2019). Specifically, Providencia alcalifaciens has been linked to several diarrheal outbreaks worldwide, raising public safety concerns (Shah et al., 2019; Shah et al., 2015; Hinenoya et al., 2009; Yoh et al., 2005). The genome of P. alcalifaciens 205/92, a clinical isolate, has been sequenced and was found to contain numerous genes with homology to many virulence factors in pathogenic Gram-negative bacteria including a type VI secretion system (T6SS). This system works as a transmembrane protein nanomachine to puncture and deliver “effector” proteins into target cells (Yang et al., 2021). T6SS are widespread in Gram-negative bacteria and have a diverse array of functions including commensalism, stress response, biofilm formation, and metal ion scavenging (Yang et al., 2021). The role and functionality of the T6SS in P. alcalifaciens has not been determined.

To better understand the conservation of the T6SS across the Providencia genus, a bioinformatic analysis was performed. The available NCBI genomes were searched using BLAST to determine those that contained the tssJ gene, which encodes for a conserved transmembrane protein of the T6SS apparatus. A BLAST search was also performed on the 12 other “core” T6SS proteins to determine conservation among Providencia species. A BLAST tutorial was additionally constructed using an R-Markdown file. This was completed to further assist in an overall understanding of the BLAST software and to make it more accessible for those without a deep understanding of bioinformatic tools. Our bioinformatic analysis suggests that the T6SS proteins are conserved throughout the Providencia genus with the most variation in the VgrG protein. It also suggests that the tssJ gene is transmitted through horizontal gene transfer due to the variability of prevalence between Providencia species.

To better understand the functional role of the T6SS in P. alcalifaciens, we constructed two gene deletion mutants, one in tssC (∆tssC) and the other in clpV (∆clpV). TssC is a structural component of the T6SS tail complex and ClpV is an ATPase required for T6SS activity. We compared P. alcalifaciens wild type bacteria with the ∆tssC and ∆clpV mutants for: (i) colonization of mammalian cells, specifically HCT116 cells, a human colonic epithelial cell line, and (ii) bacterial killing, specifically Salmonella enterica serovar Typhimurium and Escherichia coli. Our studies indicate that the T6SS in P. alcalifaciens has neither anti-prokaryotic, nor anti-eukaryotic, properties. Further research is required to determine if P. alcalifaciens uses its T6SS in other manners.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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