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.

Clinical features of SARS-CoV-2 Omicron variant infection, including incubation period and transmission rates, distinguish this variant from preceding variants. However, whether the duration of shedding of viable virus differs between omicron and previous variants is not well understood. To characterize how variant and vaccination status impact shedding of viable virus, we serially sampled symptomatic outpatients newly diagnosed with COVID-19. Anterior nasal swabs were tested for viral load, sequencing, and viral culture. Time to PCR conversion was similar between individuals infected with the Delta and the Omicron variant. Time to culture conversion was also similar, with a median time to culture conversion of 6 days (interquartile range 4-8 days) in both groups. There were also no differences in time to PCR or culture conversion by vaccination status.

SARS-CoV-2 causes acute respiratory distress that can progress to multiorgan failure and death in a minority of patients. Although severe COVID-19 disease is linked to exuberant inflammation, how SARS-CoV-2 triggers inflammation is not understood. Monocytes and macrophages are sentinel immune cells in the blood and tissue, respectively, that sense invasive infection to form inflammasomes that activate caspase-1 and gasdermin D (GSDMD) pores, leading to inflammatory death (pyroptosis) and processing and release of IL-1 family cytokines, potent inflammatory mediators. Here we show that expression quantitative trait loci (eQTLs) linked to higher GSDMD expression increase the risk of severe COVID-19 disease (odds ratio, 1.3, p<0.005). We find that about 10% of blood monocytes in COVID-19 patients are infected with SARS-CoV-2. Monocyte infection depends on viral antibody opsonization and uptake of opsonized virus by the Fc receptor CD16. After uptake, SARS-CoV-2 begins to replicate in monocytes, as evidenced by detection of double-stranded RNA and subgenomic RNA and expression of a fluorescent reporter gene. However, infection is aborted, and infectious virus is not detected in infected monocyte supernatants or patient plasma. Instead, infected cells undergo inflammatory cell death (pyroptosis) mediated by activation of the NLRP3 and AIM2 inflammasomes, caspase-1 and GSDMD. Moreover, tissue-resident macrophages, but not infected epithelial cells, from COVID-19 lung autopsy specimens showed evidence of inflammasome activation. These findings taken together suggest that antibody-mediated SARS-CoV-2 infection of monocytes/macrophages triggers inflammatory cell death that aborts production of infectious virus but causes systemic inflammation that contributes to severe COVID-19 disease pathogenesis.

Persistent SARS-CoV-2 replication and systemic dissemination are linked to increased COVID-19 disease severity and mortality. However, the precise immune profiles that track with enhanced viral clearance, particularly from systemic RNAemia, remain incompletely defined. To define whether antibody characteristics, specificities, or functions that emerge during natural infection are linked to accelerated containment of viral replication, we examined the relationship of SARS-CoV-2-specific humoral immune evolution in the setting of SARS-CoV-2 plasma RNAemia, which is tightly associated with disease severity and death. On presentation to the emergency department, S-specific IgG3, IgA1, and Fc-γ-receptor (Fcγ R) binding antibodies were all inversely associated with higher baseline plasma RNAemia. Importantly, the rapid development of spike (S) and its subunit (S1/S2/receptor binding domain)-specific IgG, especially FcγR binding activity, were associated with clearance of RNAemia. These results point to a potentially critical and direct role for SARS-CoV-2-specific humoral immune clearance on viral dissemination, persistence, and disease outcome, providing novel insights for the development of more effective therapeutics to resolve COVID-19. IMPORTANCE We showed that persistent SARS-CoV-2 RNAemia is an independent predictor of severe COVID-19. We observed that SARS-CoV-2-targeted antibody maturation, specifically Fc-effector functions rather than neutralization, was strongly linked with the ability to rapidly clear viremia. This highlights the critical role of key humoral features in preventing viral dissemination or accelerating viremia clearance and provides insights for the design of next-generation monoclonal therapeutics. The main key points will be that (i) persistent SARS-CoV-2 plasma RNAemia independently predicts severe COVID-19 and (ii) specific humoral immune functions play a critical role in halting viral dissemination and controlling COVID-19 disease progression.

Type 3 secretion systems (T3SSs) are conserved bacterial nanomachines that inject virulence proteins (effectors) into eukaryotic cells during infection. Due to their ability to inject heterologous proteins into human cells, these systems are being developed as therapeutic delivery devices. The T3SS assembles a translocon pore in the plasma membrane and then docks onto the pore. Docking activates effector secretion through the pore and into the host cytosol. Here, using Shigella flexneri, a model pathogen for the study of type 3 secretion, we determined the molecular mechanisms by which host intermediate filaments trigger docking and enable effector secretion. We show that the interaction of intermediate filaments with the translocon pore protein IpaC changed the pore's conformation in a manner that was required for docking. Intermediate filaments repositioned residues of the Shigella pore protein IpaC that are located on the surface of the pore and in the pore channel. Restricting these conformational changes blocked docking in an intermediate filament-dependent manner. These data demonstrate that a host-induced conformational change to the pore enables T3SS docking and effector secretion, providing new mechanistic insight into the regulation of type 3 secretion.

Several species of pathogenic bacteria replicate within an intracellular vacuolar niche. Bacteria that escape into the cytosol are captured by the autophagic pathway and targeted for lysosomal degradation, representing a defense against bacterial exploitation of the host cytosol. Autophagic capture of Salmonella Typhimurium occurs predominantly via generation of a polyubiquitin signal around cytosolic bacteria, binding of adaptor proteins, and recruitment of autophagic machinery. However, the components mediating bacterial target selection and ubiquitination remain obscure. We identify LRSAM1 as the E3 ligase responsible for anti-Salmonella autophagy-associated ubiquitination. LRSAM1 localizes to several intracellular bacterial pathogens and generates the bacteria-associated ubiquitin signal; these functions require LRSAM1's leucine-rich repeat and RING domains, respectively. Using cells from LRSAM1-deficient individuals, we confirm that LRSAM1 is required for ubiquitination associated with intracellular bacteria but dispensable for ubiquitination of aggregated proteins. LRSAM1 is therefore a bacterial recognition protein and ubiquitin ligase that defends the cytoplasm from invasive pathogens.

Shigella surface protein IcsA and its cytoplasmic derivatives are localized to the old pole of rod-shaped cells when expressed in Escherichia coli. In spherical mreB cells, IcsA is targeted to ectopic sites and close to one extremity of actin-like MamK filament. To gain insight into the properties of the sites containing polar material, we studied the IcsA localization in spherical cells. GFP was exported into the periplasm via the Tat pathway and used as a periplasmic space marker. GFP displayed zonal fluorescence in both mreB and rodA-pbpA spherical E. coli cells, indicating an uneven periplasmic space. Deconvolution images revealed that the cytoplasmic IcsA fused to mCherry was localized outside or at the edge of the GFP zones. These observations strongly suggest that polar material is restricted to the positions where the periplasm possesses particular structural or biochemical properties.

Many Gram-negative bacterial pathogens require a type 3 secretion system (T3SS) to deliver effector proteins into eukaryotic cells. Contact of the tip complex of the T3SS with a target eukaryotic cell initiates secretion of the two bacterial proteins that assemble into the translocon pore in the plasma membrane. The translocon pore functions to regulate effector protein secretion and is the conduit for effector protein translocation across the plasma membrane. To generate insights into how the translocon pore regulates effector protein secretion, we defined the topology of the Shigella translocon pore protein IpaC in the plasma membrane following its native delivery by the T3SS. Using single cysteine substitution mutagenesis and site-directed labeling with a membrane-impermeant chemical probe, we mapped residues accessible from the extracellular surface of the cell. Our data support a model in which the N terminus of IpaC is extracellular and the C terminus of IpaC is intracellular. These findings resolve previously conflicting data on IpaC topology that were based on nonnative delivery of IpaC to membranes. Salmonella enterica serovar Typhimurium also requires the T3SS for effector protein delivery into eukaryotic cells. Although the sequence of IpaC is closely related to the Salmonella translocon pore protein SipC, the two proteins have unique functional attributes during infection. We showed a similar overall topology for SipC and IpaC and identified subtle topological differences between their transmembrane α-helixes and C-terminal regions. Together, our data suggest that topological differences among distinct translocon pore proteins may dictate organism-specific functional differences of the T3SSs during infection.IMPORTANCE The type 3 secretion system (T3SS) is a nanomachine required for virulence of many bacterial pathogens that infect humans. The system delivers bacterial virulence proteins into the cytosol of human cells, where the virulence proteins promote bacterial infection. The T3SS forms a translocon pore in the membranes of target cells. This pore is the portal through which bacterial virulence proteins are delivered by the T3SS into the eukaryotic cytosol. The pore also regulates secretion of these virulence proteins. Our work defines the topology of translocon pore proteins in their native context during infection, resolves previously conflicting reports about the topology of the Shigella translocon pore protein IpaC, and provides new insights into how interactions of the pore with the T3SS likely produce signals that activate secretion of virulence proteins.

In the outer membrane of gram-negative bacteria, O-antigen segments of lipopolysaccharide (LPS) form a chemomechanical barrier, whereas lipid A moieties anchor LPS molecules. Upon infection, human guanylate binding protein-1 (hGBP1) colocalizes with intracellular gram-negative bacterial pathogens, facilitates bacterial killing, promotes activation of the lipid A sensor caspase-4, and blocks actin-driven dissemination of the enteric pathogen Shigella. The underlying molecular mechanism for hGBP1's diverse antimicrobial functions is unknown. Here, we demonstrate that hGBP1 binds directly to LPS and induces "detergent-like" LPS clustering through protein polymerization. Binding of polymerizing hGBP1 to the bacterial surface disrupts the O-antigen barrier, thereby unmasking lipid A, eliciting caspase-4 recruitment, enhancing antibacterial activity of polymyxin B, and blocking the function of the Shigella outer membrane actin motility factor IcsA. These findings characterize hGBP1 as an LPS-binding surfactant that destabilizes the rigidity of the outer membrane to exert pleiotropic effects on the functionality of gram-negative bacterial cell envelopes.

The type III secretion system is required for virulence of many pathogenic bacteria. Bacterial effector proteins delivered into target host cells by this system modulate host signaling pathways and processes in a manner that promotes infection. Here, we define the activity of the effector protein OspB of the human pathogen Shigella spp., the etiological agent of shigellosis and bacillary dysentery. Using the yeast Saccharomyces cerevisiae as a model organism, we show that OspB sensitizes cells to inhibition of TORC1, the central regulator of growth and metabolism. In silico analyses reveal that OspB bears structural homology to bacterial cysteine proteases that target mammalian cell processes, and we define a conserved cysteine-histidine catalytic dyad required for OspB function. Using yeast genetic screens, we identify a crucial role for the arginine N-degron pathway in the yeast growth inhibition phenotype and show that inositol hexakisphosphate is an OspB cofactor. We find that a yeast substrate for OspB is the TORC1 component Tco89p, proteolytic cleavage of which generates a C-terminal fragment that is targeted for degradation via the arginine N-degron pathway; processing and degradation of Tco89p is required for the OspB phenotype. In all, we demonstrate that the Shigella T3SS effector OspB is a cysteine protease and decipher its interplay with eukaryotic cell processes. IMPORTANCEShigella spp. are important human pathogens and among the leading causes of diarrheal mortality worldwide, especially in children. Virulence depends on the Shigella type III secretion system (T3SS). Definition of the roles of the bacterial effector proteins secreted by the T3SS is key to understanding Shigella pathogenesis. The effector protein OspB contributes to a range of phenotypes during infection, yet the mechanism of action is unknown. Here, we show that S. flexneri OspB possesses cysteine protease activity in both yeast and mammalian cells, and that enzymatic activity of OspB depends on a conserved cysteine-histidine catalytic dyad. We determine how its protease activity sensitizes cells to TORC1 inhibition in yeast, finding that OspB cleaves a component of yeast TORC1, and that the degradation of the C-terminal cleavage product is responsible for OspB-mediated hypersensitivity to TORC1 inhibitors. Thus, OspB is a cysteine protease that depends on a conserved cysteine-histidine catalytic dyad.

Sepsis, defined as organ dysfunction caused by a dysregulated host-response to infection, is characterized by immunosuppression. The vasopressor norepinephrine is widely used to treat low blood pressure in sepsis but exacerbates immunosuppression. An alternative vasopressor is angiotensin-II, a peptide hormone of the renin-angiotensin system (RAS), which displays complex immunomodulatory properties that remain unexplored in severe infection. In a murine cecal ligation and puncture (CLP) model of sepsis, we found alterations in the surface levels of RAS proteins on innate leukocytes in peritoneum and spleen. Angiotensin-II treatment induced biphasic, angiotensin-II type 1 receptor (AT1R)-dependent modulation of the systemic inflammatory response and decreased bacterial counts in both the blood and peritoneal compartments, which did not occur with norepinephrine treatment. The effect of angiotensin-II was preserved when treatment was delivered remote from the primary site of infection. At an independent laboratory, angiotensin-II treatment was compared in LysM-Cre AT1aR-/- (Myeloid-AT1a-) mice, which selectively do not express AT1R on myeloid-derived leukocytes, and littermate controls (Myeloid-AT1a+). Angiotensin-II treatment significantly reduced post-CLP bacteremia in Myeloid-AT1a+ mice but not in Myeloid-AT1a- mice, indicating that the AT1R-dependent effect of angiotensin-II on bacterial clearance was mediated through myeloid-lineage cells. Ex vivo, angiotensin-II increased post-CLP monocyte phagocytosis and ROS production after lipopolysaccharide stimulation. These data identify a mechanism by which angiotensin-II enhances the myeloid innate immune response during severe systemic infection and highlight a potential role for angiotensin-II to augment immune responses in sepsis.

Endotoxin-bacterial lipopolysaccharide (LPS)-is a driver of lethal infection sepsis through excessive activation of innate immune responses. When delivered to the cytosol of macrophages, cytosolic LPS (cLPS) induces the assembly of an inflammasome that contains caspases-4/5 in humans or caspase-11 in mice. Whereas activation of all other inflammasomes is triggered by sensing of pathogen products by a specific host cytosolic pattern recognition receptor protein, whether pattern recognition receptors for cLPS exist has remained unclear, because caspase-4, caspase-5, and caspase-11 bind and activate LPS directly in vitro. Here, we show that the primate-specific protein NLRP11 is a pattern recognition receptor for cLPS that is required for efficient activation of the caspase-4 inflammasome in human macrophages. In human macrophages, NLRP11 is required for efficient activation of caspase-4 during infection with intracellular Gram-negative bacteria or upon electroporation of LPS. NLRP11 could bind LPS and separately caspase-4, forming a high-molecular weight complex with caspase-4 in HEK293T cells. NLRP11 is present in humans and other primates but absent in mice, likely explaining why it has been overlooked in screens looking for innate immune signaling molecules, most of which have been carried out in mice. Our results demonstrate that NLRP11 is a component of the caspase-4 inflammasome activation pathway in human macrophages.

There is increasing evidence that the risk of SARS-CoV-2 infection among vaccinated individuals is variant-specific, suggesting that protective immunity against SARS-CoV-2 may differ by variant. We enrolled vaccinated (n = 39) and unvaccinated (n = 11) individuals with acute, symptomatic SARS-CoV-2 Delta or Omicron infection and performed SARS-CoV-2 viral load quantification, whole-genome sequencing, and variant-specific antibody characterization at the time of acute illness and convalescence. Viral load at the time of infection was inversely correlated with antibody binding and neutralizing antibody responses. Increases in antibody titers and neutralizing activity occurred at convalescence in a variant-specific manner. Across all variants tested, convalescent neutralization titers in unvaccinated individuals were markedly lower than in vaccinated individuals. For individuals infected with the Delta variant, neutralizing antibody responses were weakest against BA.2, whereas infection with Omicron BA.1 variant generated a broader response against all tested variants, including BA.2.

Protective immunity against SARS-CoV-2 infection after COVID-19 vaccination may differ by variant. We enrolled vaccinated (n = 39) and unvaccinated (n = 11) individuals with acute, symptomatic SARS-CoV-2 Delta or Omicron infection and performed SARS-CoV-2 viral load quantification, whole-genome sequencing, and variant-specific antibody characterization at the time of acute illness and convalescence. Viral load at the time of infection was inversely correlated with antibody binding and neutralizing antibody responses. Across all variants tested, convalescent neutralization titers in unvaccinated individuals were markedly lower than in vaccinated individuals. Increases in antibody titers and neutralizing activity occurred at convalescence in a variant-specific manner. For example, among individuals infected with the Delta variant, neutralizing antibody responses were weakest against BA.2, whereas infection with Omicron BA.1 variant generated a broader response against all tested variants, including BA.2.

Type 3 secretion systems (T3SSs) of bacterial pathogens translocate bacterial effector proteins that mediate disease into the eukaryotic cytosol. Effectors traverse the plasma membrane through a translocon pore formed by T3SS proteins. In a genome-wide selection, we identified the intermediate filament vimentin as required for infection by the T3SS-dependent pathogen S. flexneri. We found that vimentin is required for efficient T3SS translocation of effectors by S. flexneri and other pathogens that use T3SS, Salmonella enterica serovar Typhimurium and Yersinia pseudotuberculosis. Vimentin and the intestinal epithelial intermediate filament keratin 18 interact with the C-terminus of the Shigella translocon pore protein IpaC. Vimentin and its interaction with IpaC are dispensable for pore formation, but are required for stable docking of S. flexneri to cells; moreover, stable docking triggers effector secretion. These findings establish that stable docking of the bacterium specifically requires intermediate filaments, is a process distinct from pore formation, and is a prerequisite for effector secretion.

The introduction of vaccines has inspired hope in the battle against SARS-CoV-2. However, the emergence of viral variants, in the absence of potent antivirals, has left the world struggling with the uncertain nature of this disease. Antibodies currently represent the strongest correlate of immunity against SARS-CoV-2, thus we profiled the earliest humoral signatures in a large cohort of acutely ill (survivors and nonsurvivors) and mild or asymptomatic individuals with COVID-19. Although a SARS-CoV-2–specific immune response evolved rapidly in survivors of COVID-19, nonsurvivors exhibited blunted and delayed humoral immune evolution, particularly with respect to S2-specific antibodies. Given the conservation of S2 across β-coronaviruses, we found that the early development of SARS-CoV-2–specific immunity occurred in tandem with preexisting common β-coronavirus OC43 humoral immunity in survivors, which was also selectively expanded in individuals that develop a paucisymptomatic infection. These data point to the importance of cross-coronavirus immunity as a correlate of protection against COVID-19.

Mucormycosis involves life-threatening rapidly progressive angioinvasion with infiltration across tissue planes, resulting in necrosis and thrombosis, most commonly seen in the setting of immunocompromised states. We describe 2 cases of isolated cerebral mucormycosis in immunocompetent adults and describe this syndrome in detail in the context of a systemic literature review.

During infection, some bacterial pathogens invade the eukaryotic cytosol and spread between cells of an epithelial monolayer. Intercellular spread occurs when these pathogens push against the plasma membrane, forming protrusions that are engulfed by adjacent cells. Here, we show that IpaC, a Shigella flexneri type 3 secretion system protein, binds the host cell-adhesion protein β-catenin and facilitates efficient protrusion formation. S. flexneri producing a point mutant of IpaC that cannot interact with β-catenin is defective in protrusion formation and spread. Spread is restored by chemical reduction of intercellular tension or genetic depletion of β-catenin, and the magnitude of the protrusion defect correlates with membrane tension, indicating that IpaC reduces membrane tension, which facilitates protrusion formation. IpaC stabilizes adherens junctions and does not alter β-catenin localization at the membrane. Thus, Shigella, like other bacterial pathogens, reduces intercellular tension to efficiently spread between cells.

Shigella spp. are intracytosolic gram-negative pathogens that cause disease by invasion and spread through the colonic mucosa, utilizing host cytoskeletal components to form propulsive actin tails. We have previously identified the host factor Toca-1 as being recruited to intracellular S. flexneri and being required for efficient bacterial actin tail formation. We show that at early times during infection (40 min.), the type three-secreted effector protein IcsB recruits Toca-1 to intracellular bacteria and that recruitment of Toca-1 is associated with repression of recruitment of LC3, as well as with repression of recruitment of the autophagy marker NDP52, around these intracellular bacteria. LC3 is best characterized as a marker of autophagosomes, but also marks phagosomal membranes in the process LC3-associated phagocytosis. IcsB has previously been demonstrated to be required for S. flexneri evasion of autophagy at late times during infection (4-6 hr) by inhibiting binding of the autophagy protein Atg5 to the Shigella surface protein IcsA (VirG). Our results suggest that IcsB and Toca-1 modulation of LC3 recruitment restricts LC3-associated phagocytosis and/or LC3 recruitment to vacuolar membrane remnants. Together with published results, our findings suggest that IcsB inhibits innate immune responses in two distinct ways, first, by inhibiting LC3-associated phagocytosis and/or LC3 recruitment to vacuolar membrane remnants early during infection, and second, by inhibiting autophagy late during infection.

The intracellular bacterial pathogen Shigella infects and spreads through the human intestinal epithelium. Effector proteins delivered by Shigella into cells promote infection by modulating diverse host functions. We demonstrate that the effector protein OspB interacts directly with the scaffolding protein IQGAP1, and that the absence of either OspB or IQGAP1 during infection leads to larger areas of S. flexneri spread through cell monolayers. We show that the effect on the area of bacterial spread is due to OspB triggering increased cell proliferation at the periphery of infected foci, thereby replacing some of the cells that die within infected foci and restricting the area of bacterial spread. We demonstrate that OspB enhancement of cell proliferation results from activation of mTORC1, a master regulator of cell growth, and is blocked by the mTORC1-specific inhibitor rapamycin. OspB activation of mTORC1, and its effects on cell proliferation and bacterial spread, depends on IQGAP1. Our results identify OspB as a regulator of mTORC1 and mTORC1-dependent cell proliferation early during S. flexneri infection and establish a role for IQGAP1 in mTORC1 signaling. They also raise the possibility that IQGAP1 serves as a scaffold for the assembly of an OspB-mTORC1 signaling complex.