Shigella flexneri Regulation of ARF6 Activation during Bacterial Entry via an IpgD-Mediated Positive Feedback Loop

ABSTRACT Entry into cells is critical for virulence of the human bacterial pathogens Shigella spp. Shigella spp. induce membrane ruffle formation and macropinocytic uptake, but the events instigating this process are incompletely understood. The host small GTPase ADP-ribosylation factor 6 (ARF6) functions in membrane trafficking at the plasma membrane and activates membrane ruffle formation. We demonstrate that ARF6 is required for efficient Shigella flexneri entry, is activated by S. flexneri dependent on the phosphatase activity of the type III secreted effector IpgD, and depends on cytohesin guanine nucleotide exchange factors (GEFs) for recruitment to entry sites. The cytohesin GEF ARF nucleotide binding site opener (ARNO) is recruited to these sites, also dependent on IpgD phosphatase activity. ARNO recruitment is independent of ARF6, indicating that, in addition to the described recruitment of ARNO by ARF6, ARNO is recruited upstream of ARF6. Our data provide evidence that ARF6, IpgD, phosphoinositide species, and ARNO constitute a previously undescribed positive feedback loop that amplifies ARF6 activation at bacterial entry sites, thereby promoting efficient S. flexneri uptake

Characterization of host and bacterial factors critical for Shigella flexneri pathogenesis

Shigella species are Gram-negative bacterial pathogens acquired by fecal-oral spread. A common cause of bacillary dysentery worldwide, particularly in developing countries, Shigella invade colonic mucosal cells and employ a repertoire of proteins secreted by a type III secretion system (T3SS) to manipulate host cytoskeleton and signaling pathways. The bacterium escapes from the vacuole, enters the host cell cytoplasm, evades host recognition, and spreads intercellularly. In this thesis, I describe two independent projects that aim to clarify the complex interplay between bacterial effectors and host processes critical to Shigella flexneri pathogenesis. Primarily, I examined the role of host small GTPase ADP-ribosylation factor 6 (ARF6) in S. flexneri entry. ARF6 functions in membrane trafficking at the plasma membrane and activates membrane ruffle formation. I show here that ARF6 is required for efficient entry, and together with the host guanine nucleotide exchange factor (GEF) ARF nucleotide binding site opener (ARNO) and the T3SS effector IpgD, constitutes a positive feedback loop that amplifies ARF6 activation at S. flexneri entry sites to promote efficient bacterial uptake. I also investigated the outer Shigella protein C (OspC) effectors OspC1, OspC2, and OspC3, highly homologous T3SS effectors with potentially disparate functions. Preliminary evidence suggested that OspC2 and/or OspC3 interact with host protein Toca-1, which is implicated in actin polymerization and pathogen recognition by host innate immune processes. My evidence suggests the OspC proteins are important for evasion of autophagy and/or other mechanisms of intracellular survival and replication. This work identifies new cellular targets and activities of the Shigella effector IpgD and provides insight into mechanisms of pathogenesis dependent on secreted bacterial effectors. The lines of investigation presented here demonstrate the intricate ways in which S. flexneri both exploits and subverts host processes in order to survive in the intracellular environment

The Koebner phenomenon may contribute to the development of calciphylaxis: A case series

Calciphylaxis is characterized by calcific occlusion of vessels and subsequent tissue ischemia due to thrombosis. The precise pathogenetic mechanism behind calciphylaxis remains unclear. In the original experiment by Hans Selye and colleagues, soft-tissue calcification was induced in rats by applying a sensitizing agent, followed by a “challenger” agent after a specific time period. Trauma may represent one of these “challenger” agents, serving as an inducer of endothelial dysfunction and subsequent thrombosis, leading from tissue calcification to calciphylaxis. Koebnerization, a term used to describe the appearance of isomorphic lesions in areas of trauma, has been postulated to be a feature of calciphylaxis. This hypothesis arose from reports of patients who developed calciphylaxis following mild skin trauma, such as that caused by chronic resting of elbows on thighs, placement of ice packs, and injections involving various medications such as iron dextran, tobramycin, and especially insulin. Rigorous studies demonstrating the relationship between calciphylaxis and Koebnerization and an underlying mechanism are limited. To better understand this association, this study retrospectively identified characteristics of patients who presented with calciphylaxis in areas of trauma, suggesting the presence of Koebnerization

Penile calciphylaxis: A retrospective case-control study

BACKGROUND: Calciphylaxis is a rare disorder characterized by skin necrosis caused by calcium deposition within vessels, thrombosis, and subsequent tissue ischemia. Penile involvement may rarely occur. OBJECTIVE: To identify risk factors, diagnosis, management, and mortality of patients with penile calciphylaxis. METHODS: A retrospective medical record review was conducted of 16 patients with penile calciphylaxis treated at 2 large urban tertiary care centers between January 2001 and December 2019. A control group of 44 male patients with nonpenile calciphylaxis at the same institution was included. RESULTS: The median survival of patients with penile calciphylaxis was 3.8 months (interquartile range, 27.0 months). Mortality was 50% at 3 months and 62.5% at 6 months for penile calciphylaxis, and 13.6% at 3 months and 29.5% at 6 months for controls (P = .008). Patients with penile calciphylaxis were less likely to be obese (P = .04) but more likely to have hyperparathyroidism (P = .0003) and end-stage renal disease (P = .049). LIMITATIONS: Retrospective study design and small sample size. CONCLUSIONS: This study further defines the disease course of penile calciphylaxis, which has high mortality. Imaging may be used to aid diagnosis. Risk factors include end-stage renal disease, hyperparathyroidism, and normal body mass index