Department of Biological Sciences

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Author: search.filters.author.Marathe, SandhyaSubject: search.filters.subject.Caenorhabditis elegans

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    Characterizing the type 6 secretion system (T6SS) of E. cloacae SBP-8 and its role in pathogenesis and bacterial competition
    (Elsevier, 2023-10) Jha, Prabhat Nath; Marathe, Sandhya
    Despite the relevance of E. cloacae as an opportunistic pathogen, very little is known about its pathogenicity mechanism and the factors influencing its virulence. The mechanism of E. cloacae pathogenicity appears to be complex and multifactorial, with the presence of different putative virulence factors whose role is still not clear in the development of the disease. In this study, we systematically investigated the role of T6SS (type six secretion system) of E. cloacae SBP-8, an environmental isolate, in eukaryotic and bacterial cell interaction. Analysis of the genome sequence of E. cloacae SBP-8 revealed the presence of sets of genes coding for the expression of one complete T6SS cluster, which is similar to T6SS-1 cluster of E. cloacae ATCC 13047 (clinical isolates). In addition, an Hcp effector protein was detected in the secretome, and this secretion depended on ClpV, an Atpase of T6SS, confirming that strain SBP-8 produces functional T6SS. Deletion of T6SS-associated gene clpV did not induce any significant change in the life span and rate of colonization in C. elegans. No major significant change was observed in the expression profiling of antimicrobial genes (clec-60, clec-85, clec-87 and lys-1) and toll-like receptor (toll-1) gene, involved in stimulating an immune response against the pathogen. No difference in the ability to invade and proliferate in intestinal cells and phagocytosis by macrophages was observed. In addition, we demonstrated that the ability of E. cloacae SBP-8 to out-compete Escherichia coli was reliant upon its T6SS in contact-dependent manner. Our results show that T6SS of the environmental isolates is required for interbacterial competition but not for invasion and proliferation inside host cells.
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    Elucidating the pathogenic potential of Enterobacter cloacae SBP-8 using Caenorhabditis elegans as a model host
    (Elsiever, 2020-11) Jha, Prabhat N.; Marathe, Sandhya
    Enterobacter cloacae, an opportunistic nosocomial pathogen, is reported to possess different virulence factors that could potentially influence its pathogenesis. Generally, the E. cloacae infections are of endogenous origin occurring in immunocompromised patients. The mechanisms of pathogenicity remain elusive, possibly due to the absence of established model hosts. Thus, we explored the utility of Caenorhabditis elegans as a model host to test the pathogenicity of E. cloacae SBP-8, a soil isolate. E. cloacae SBP-8 progressively colonized the intestine of C. elegans. It induced cell death (as assessed through DNA damage), reproductive defect and reduction of lifespan, comparable to a clinical isolate, E. cloacae (MTCC 509). Observation with Nomarski microscopy revealed significant anterior pharyngeal distention, and altered egg arrangement with internal egg hatching in 70% infected worms. The internal egg hatching was observed as early as 48 h post infection. E. cloacae SBP-8 infection reduced the brood size by 16%. A 2′,7′-dichlorodihydrofluorescein diacetate staining confirmed the 10-fold induction of reactive oxygen species implicating either mitochondrial damage or septic shock in infected worms. Expression analysis through RT-PCR indicated stimulation of immune response by E. cloacae SBP-8 in worms by upregulating tol-1, a Toll-like receptor, within 6 h of exposure. During the initial phase of infection (up to 24 h) the nematodes exhibited protective immune response by upregulating antimicrobial peptide genes, lys-1, clec-60, clec-85, and clec-87. However, these genes were downregulated at later hours (48 h), indicating the nematodes surrendered to the infection. A similar trend was observed for reproductive genes (lin-29 and let-23), suggesting a struggle to maintain functional reproduction by the nematodes. These results clearly demonstrate the pathogenic potential of E. cloacae SBP-8 and suggest the suitability of C. elegans as a model organism to study its pathogenesis. This is the first study indicating that E. cloacae infections could potentially originate from an exogenic source (here soil).