SalmonellaTyphimurium outer membrane protein A (OmpA) renders protection against nitrosative stress by promoting SCV stability in murine macrophages

Abstract

Porins are highly conserved bacterial outer membrane proteins having β barrel structure and are majorly involved in the selective transport of charged molecules across the membrane. Despite having huge contributions in the pathogenesis of many gram-negative bacteria, their role remains elusive in salmonellosis. In this study, we have characterized the pathogenic role of porins majorly found on the outer membrane of Salmonella Typhimurium (such as OmpA, OmpC, OmpD, and OmpF) paying the utmost importance to OmpA. The outer membrane protein A (OmpA) of Salmonella Typhimurium has shown a multifaceted role in our study. We have observed that deletion of ompA from wildtype Salmonella has made it more prone to phagocytosis and weakly proliferative in macrophages. Whereas, in epithelial cells STM ΔompA was found to be invasion deficient and hyper-proliferative. The poor colocalization of STM ΔompA with LAMP-1 confirmed impaired stability of SCV membrane around the intracellular bacteria, which further resulted in the release of the knockout strain to the cytosol of macrophage where it is bombarded with reactive nitrogen intermediates (RNI). The cytosolic localization of STM ΔompA was found to be responsible for the downregulation of SPI-2 encoded virulent factor SpiC which is required for suppressing the activity of iNOS. The reduced recruitment of nitrotyrosine on wildtype Salmonella staying in the cytosol of macrophage by ectopically expressing Listeriolysin O (LLO) strongly proves the pro-bacterial role of OmpA against host nitrosative stress. The time-dependent increase in 405/ 488 ratio of STM ΔompA pQE60-Grx1-roGFP2 exposed to in vitro acidified nitrite suggested RNI dependent redox burst which further answered the reason for its enhanced sensitivity towards nitrosative stress. Our study further demonstrated loss of integrity and enhanced porosity in the bacterial outer membrane in absence of OmpA. The enhanced porosity of the bacterial outer membrane was further attributed to the upregulated expression of larger porins namely ompC, ompD, and ompF. In comparison with STM ΔompA ΔompC and ΔompA ΔompF, the enhanced uptake of nitrite and greater recruitment of nitrotyrosine on intracellular STM ΔompA ΔompD demonstrate the involvement of OmpC and OmpF in the entry of excess nitrite in ompA deficient bacteria.

The extracellular loops of Salmonella Typhimurium outer membrane protein A (OmpA) maintain the stability of Salmonella containing vacuole (SCV) in murine macrophages and protect the bacteria from autophagy-dependent lysosomal degradation

“…Earlier, our group has shown that this cytosolic population of the bacteria becomes hyper-proliferative inside the epithelial cells, and their intracellular proliferation was significantly attenuated in macrophages. Our study further depicted that STM ΔompA is unable to recruit T3SS2 translocon proteins (SseC and SseD) on its surface and is deficient in producing an SPI-2 effector [15]. Available literature suggests that Salmonella deficient in making active T3SS2 and with damaged SCV cannot suppress host autophagy machinery.…”

“…Earlier, we have shown an enhanced expression of ompA transcript in wild type Salmonella Typhimurium growing intracellularly in murine macrophages at 9 th and 12 th hours postinfection, which suggested the requirement of OmpA for the intracellular survival of bacteria [15]. Our current study revealed that in the absence of OmpA, Salmonella cannot stay inside SCV and comes into the cytosol of host cells.…”

“…Most of the studies that have addressed the intracellular behavior of Salmonella so far have discovered the role of SPI-1 and SPI-2 encoded type 3 secretion systems (T3SS1 and T3SS2) and virulent factors in SCV biogenesis [17]. Earlier, our group has reported the role of outer membrane protein A in maintaining the stability of SCV within murine macrophages [15]. To validate our previous observation, the vacuolar niche of the wild type, ompA deficient, and complemented strains of S. Typhimurium in murine macrophages was checked.…”

“…Wild type Salmonella can recruit non-receptor tyrosine kinase or focal adhesion kinase on the surface of the SCV in an SPI-2 encoded T3SS2 dependent manner to suppress the host autophagy machinery by activating the Akt-mTOR signaling pathway [18, 19]. A previous study from our group proved that intracellular STM ΔompA is unable to produce and secrete the SPI-2 encoded translocon proteins into the host cell’s cytosol [15], suggesting the formation of a malfunctioning T3SS2. Hence, we have hypothesized that the infection of murine macrophages with STM ΔompA may activate host autophagy machinery.…”

“…The deficiency of OmpA compromises the biofilm-forming ability of the bacteria in response to bile salt stress [14]. In our previous study, we have proved that deletion of OmpA significantly hampers the stability of the outer membrane of Salmonella , which makes the bacteria susceptible to in vitro and in vivo nitrosative stress[15]. We have also found that ompA knockout Salmonella quits the SCV during the late phase of infection in murine macrophages, suggesting a previously unknown role of OmpA in maintaining the stability of SCV.…”