Hensel, M. | bioRxiv Science

bioRxiv2019

Structural and functional characterisation of SiiA, an auxiliary protein from the SPI4-encoded type 1 secretion system from Salmonella enterica

Salmonella invasion is mediated by a concerted action of the Salmonella pathogenicity island 4 (SPI4)-encoded type one secretion system (T1SS) and the SPIl-encoded type three secretion system (T3SS-1). The SPI4-encoded T1SS establishes the first contact to the host membrane. It consists of five proteins (SiiABCDF) that secrete the giant adhesin SiiE. The exact mechanism by which the T1SS enables host cell recognition remains unclear. Here, we investigated structure-function relationships in SiiA, a non-canonical T1SS subunit located at the inner membrane (IM). We observe that SiiA consists of a membrane domain, an intrinsically disordered periplasmic linker region and a folded globular periplasmic domain (SiiA-PD). The crystal structure of SiiA-PD shows homology to that of MotB-PD and other peptidoglycan (PG)-binding domains. Indeed, SiiA-PD binds PG in vitro albeit at an acidic pH, only, whereas MotB-PD binds PG from pH 5.8 to 8. Mutation of Arg162 in SiiA impedes PG binding and reduces Salmonella invasion efficacy of polarized epithelial cells. SiiA forms a complex with SiiB at the IM, and the SiiA-MotB homology is likely paralleled by a SiiB-MotA homology. We show that, in addition to PG binding, the SiiAB complex translocates protons across the IM. Substituting Asp13 in SiiA impairs proton translocation. Overall, SiiA displays many properties previously observed in MotB. However, whereas the MotAB complex uses the proton motif force (PMF) to energize the bacterial flagellum, it remains to be shown how the use of the PMF by SiiAB assists T1SS function and ultimately Salmonella invasion.

microbiology↗

bioRxiv2018

Characterization of a novel intratracheal aerosol challenge model of Brucella melitensis in guinea pigs

B. melitensis is considered the most virulent of the Brucella species, and a need exists for an improved laboratory animal model of infection that mimics natural transmission and disease. Guinea pigs are highly susceptible to infection with Brucella spp. and develop a disease syndrome that mimics natural disease after aerosol inoculation. Intratracheal inoculation is a targeted means of generating aerosols that offer advantages over aerosol chamber delivery. To establish this delivery method, female, Hartley guinea pigs were infected via intratracheal inoculation with PBS or 16M B. melitensis at low dose (101 to 103) or high dose (106 to 108) and monitored for 30 days for signs of disease. Guinea pigs in the high dose groups developed fever between 12-17 days post-inoculation. Bacteria were recovered from the spleen, liver, lymph nodes, lung, and uterus at 30-days post-inoculation and demonstrated dose dependent mean increases in colonization and pathologic changes consistent with human brucellosis. To study the kinetics of extrapulmonary dissemination, guinea pigs were inoculated with 107 CFU and euthanized at 2-hours post inoculation and at weekly intervals for 3 weeks. 5.8x105 to 4.2x106 CFU were recovered from the lung 2 hours post-inoculation indicating intratracheal inoculation is an efficient means of infecting guinea pigs. Starting at 1-week post inoculation bacteria were recovered from the aforementioned organs with time dependent mean increases in colonization. This data demonstrates that guinea pigs develop a disease syndrome that models the human manifestation of brucellosis, which makes the guinea pig a valuable model for pathogenesis studies.\n\nAuthor summaryBrucellosis is caused by a gram-negative, intracellular bacterial pathogen with a worldwide distribution and affects up to half a million people per year. It is a neglected zoonosis that impacts not only animal welfare, but also exert economic pressure on afflicted individuals through loss of wages and decreased productivity. In people, recurrent fever, malaise, and anorexia accompanied by enlargement of the spleen and lymph nodes are common clinical symptoms of infection. The mouse model has been used extensively to study the pathogenesis of brucellosis, but there are drawbacks to extrapolating studies in mice to develop vaccines or therapeutics for people. Mice are frequently inoculated via intraperitoneal injection, which is an artificial means of producing disease that does not mimic natural transmission or disease features, such as fever. An animal model is needed that can be infected through natural transmission routes and subsequently develop a syndrome that matches clinical disease seen in people in order to study the pathogenesis of disease and to develop vaccines and therapeutics. The guinea pig offers an improvement on the mouse model because it can be infected via aerosol inoculation and develops fever, a humoral immune response, systemic colonization, and macroscopic and microscopic lesions of disease. As such, guinea pigs could be used a more biologically relevant model for evaluation of host-pathogen interactions.

microbiology↗