A novel rspA gene regulates biofilm formation and virulence of Salmonella Typhimurium.

Salmonella spp. are facultative anaerobic, Gram-negative, rod-shaped bacteria and belongs to the Enterobacteriaceae family. Although much has been known about Salmonella pathogenesis, the functional characterizations of certain genes are yet to be explored. The rspA (STM14_1818) is one such gene with putative dehydratase function, and its role in pathogenesis is unknown. The background information showed that rspA gene is upregulated in Salmonella when it resides inside macrophages, which led us to investigate its role in Salmonella pathogenesis. We generated the rspA knockout strain and complement strain in S. Typhimurium 14028. Ex-vivo and in-vivo infectivity was looked at macrophage and epithelial cell lines and Caenorhabditis elegans (C. elegans). The mutant strain differentially formed the biofilm at different temperatures by altering the expression of genes involved in the synthesis of cellulose and curli. Besides, the mutant strain is hyperproliferative intracellularly and showed increased bacterial burden in C. elegans. The mutant strain became more infectious and lethal, causing faster death of the worms than the wild type, and also modulates the worm's innate immunity. Thus, we found that the rspA deletion mutant was more pathogenic. In this study, we concluded that the rspA gene differentially regulates the biofilm formation in a temperature dependent manner by modulating the genes involved in the synthesis of cellulose and curli and negatively regulates the Salmonella virulence for longer persistence inside the host.

Immune modulations and survival strategies of evolved hypervirulent Salmonella Typhimurium strains.

BACKGROUND: Evolving multidrug-resistance and hypervirulence in Salmonella is due to multiple host-pathogen, and non-host environmental interactions. Previously we had studied Salmonella adaptation upon repeated exposure in different in-vitro and in-vivo environmental conditions. This study deals with the mechanistic basis of hypervirulence of the passaged hypervirulent Salmonella strains reported previously. METHODS: Real-time PCR, flow cytometry, western blotting, and confocal microscopy were employed to check the alteration of signaling pathways by the hypervirulent strains. The hypervirulence was also looked in-vivo in the Balb/c murine model system. RESULTS: The hypervirulent strains altered cytokine production towards anti-inflammatory response via NF-κB and Akt-NLRC4 signaling in RAW-264.7 and U-937 cells. They also impaired lysosome number, as well as co-localization with the lysosome as compared to unpassaged WT-STM. In Balb/c mice also they caused decreased antimicrobial peptides, reduced nitric oxide level, altered cytokine production, and reduced CD4+ T cell population leading to increased organ burden. CONCLUSIONS: Hypervirulent Salmonella strains infection resulted in an anti-inflammatory environment by upregulating IL-10 and down-regulating IL-1ß expression. They also evaded lysosomal degradation for their survival. With inhibition of NF-κB and Akt signaling, cytokine expression, lysosome number, as well as the bacterial burden was reverted, indicating the infection mediated immune modulation by the hypervirulent Salmonella strains through these pathways. GENERAL SIGNIFICANCE: Understanding the mechanism of adaptation can provide better disease prognosis by either targeting the bacterial gene or by strengthening the host immune system that might ultimately help in controlling salmonellosis.

Stress-induced adaptations in Salmonella: A ground for shaping its pathogenesis.

Microorganisms are able to adapt to multiple adverse environmental conditions that facilitate their survival. These microorganisms including bacteria, viruses, algae, fungi, and protozoans are exposed to different abiotic and biotic challenges throughout their life. Adaptations help these organisms to overcome the challenges and evolve as successful pathogens which at the same time might lead to severe disease outcome. The intracellular gram-negative pathogen Salmonella, the causative agent of typhoid fever has evolved into a successful pathogen and shows increasing host mortality and morbidity every year across the globe. Salmonella adapts itself in the different extreme host and non-host environments both at genetic and phenotypic level leading to their better survival and propagation. The uncontrolled and improper use of antibiotics against several Salmonella serovars has not only given rise to various multidrug resistance strains but also the emergence of hyper-infectious Salmonella strains adds to the severity of disease manifestation and treatment. Besides, several disadvantages in the existing Salmonella vaccines stand against the current therapeutic interventions against the bug. This review deals with the wide array of stresses that Salmonella encounter in its life cycle and outlines the adaptations occurring in Salmonella upon exposure to such stresses as well as how adaptations help the pathogen to withstand such extreme conditions. Insights in these aspects will help to understand Salmonella pathogenesis and associated consequences which might help in the development of new strategies in combating Salmonella infection.

Repeated in-vitro and in-vivo exposure leads to genetic alteration, adaptations, and hypervirulence in Salmonella.

Adaptation is an important phenomenon for the survival of any organism in various in-vitro and in-vivo conditions leading to their better survival. We studied Salmonella, for the evolutionary adaptation under different conditions. When Salmonella was passaged for 12 rounds in Luria Bertani (P12-STM) media, F media (F12-STM) and Caenorhabditis elegans (Ce12-STM), we found certain phenotypic variations with respect to unpassaged strain. The passaged strains showed a significant increase in motility, body size and number of flagella they possessed, increased tolerance to ROS and RNS under in-vitro condition. They were more invasive in the human intestinal epithelial cells (Int-407) and showed a higher proliferation rate in the phagocytic cells (U-937 and RAW-264.7) suggesting their efficient intracellular proliferation. The evolved strains were hyper-infectious and reduced host viability significantly in C. elegans. The upregulation of various virulent and regulatory genes observed could be associated with the evolved adaptive virulent phenotype by Salmonella in a given condition. Whole genome sequencing also indicates the genetic alteration including insertion and deletions, single nucleotide polymorphisms which are common in all the strains as well as exposure specific, giving varied phenotype among the passaged strains. This study indicates that Salmonella is showing adaptation, genetic alteration and have become hypervirulent hence is capable of causing disease severity.