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The cytosol of eukaryotic host cells is an intrinsically hostile environment for bacteria. Understanding how cytosolic pathogens adapt to and survive in the cytosol is critical to developing novel therapeutic interventions for these pathogens. The cytosolic pathogen Listeria monocytogenes requires glmR (previously known as yvcK), a gene of unknown function, for resistance to cell wall stress, cytosolic survival, inflammasome avoidance and ultimately virulence in vivo. A genetic suppressor screen revealed that blocking utilization of UDP-GlcNAc by a non-essential wall teichoic acid decoration pathway restored resistance to cell wall stress and partially restored virulence of ΔglmR mutants. In parallel, metabolomics revealed that ΔglmR mutants are impaired in the production of UDP-GlcNAc, an essential peptidoglycan and wall teichoic acid (WTA) precursor. We next demonstrated that purified GlmR can directly catalyze the synthesis of UDP-GlcNAc from GlcNAc-1P and UTP, suggesting that it is an accessory uridyltransferase. Biochemical analysis of GlmR orthologues suggest that uridyltransferase activity is conserved. Finally, mutational analysis resulting in a GlmR mutant with impaired catalytic activity demonstrated that uridyltransferase activity was essential to facilitate cell wall stress responses and virulence in vivo. Taken together these studies indicate that GlmR is an evolutionary conserved accessory uridyltransferase required for cytosolic survival and virulence of L. monocytogenes.ImportanceBacterial pathogens must adapt to their host environment in order to cause disease. The cytosolic bacterial pathogen Listeria monocytogenes requires a highly conserved protein of unknown function, GlmR (previously known as YvcK) to survive in the host cytosol. GlmR is important for resistance to some cell wall stresses and is essential for virulence. The ΔglmR mutant is deficient in production of an essential cell wall metabolite, UDP-GlcNAc, and suppressors which increase metabolite levels also restore virulence. Purified GlmR can directly catalyze the synthesis of UDP-GlcNAc and this enzymatic activity is conserved in pathogens from Firmicutes and Actinobacteria phyla. These results highlight the importance accessory cell wall metabolism enzymes in responding to cell wall stress in a variety of bacterial pathogens.

Wound repair is controlled temporally and spatially to restore tissue homeostasis. Previously we reported that thermal damage of the larval zebrafish fin disrupts collagen organization and wound healing compared to tail transection (LeBert et al., 2018). Here we characterize different injury models in larval zebrafish to dissect temporal and spatial dynamics of repair in complex damage. We found that each damage model triggers distinct inflammatory and tissue responses, with Stat3 and TGFβ playing key roles in the regulation of mesenchymal cells during simple repair. While thermal injury disrupts collagen fibers initially, healing is recovered as inflammation resolves, and mesenchymal cells and collagen fibers align. By contrast, infected wounds lead to persistent inflammation and loss of mesenchymal cells, resulting in minimal tissue repair. These wound models have broad physiological relevance, thereby providing a valuable advance in our toolkit to probe the dynamics of inflammation and wound repair in complex tissue damage.

The inflammasome is an innate immune complex whose rapid inflammatory outputs play a critical role in controlling infection; however, the host cells that mediate inflammasome responses in vivo are not well defined. Using zebrafish larvae, we examined the cellular immune responses to inflammasome activation during infection. We compared the host responses with two Listeria monocytogenes strains: wild type and Lm-pyro, a strain engineered to activate the inflammasome via ectopic expression of flagellin. Infection with Lm-pyro led to activation of the inflammasome, macrophage pyroptosis and ultimately attenuation of virulence. Depletion of caspase A, the zebrafish caspase-1 homolog, restored Lm-pyro virulence. Inflammasome activation specifically recruited macrophages to infection sites, whereas neutrophils were equally recruited to wild type and Lm-pyro infections. Similar to caspase A depletion, macrophage deficiency rescued Lm-pyro virulence to wild-type levels, while defective neutrophils had no specific effect. Neutrophils were, however, important for general clearance of L. monocytogenes, as both wild type and Lm-pyro were more virulent in larvae with defective neutrophils. This study characterizes a novel model for inflammasome studies in an intact host, establishes the importance of macrophages during inflammasome responses and adds importance to the role of neutrophils in controlling L. monocytogenes infections.

Immune cells sense and react to a multitude of factors including both host and microbe-derived signals. Understanding how cells translate these cues into particular cellular behaviors is a complex yet critical area of study. We have previously shown that both neutrophils and macrophages are important for controlling the fish pathogen Streptococcus iniae. Here, we report both host and bacterial determinants leading to the formation of organized macrophage aggregates as part of the host inflammatory response in a subset of infected larvae. Streptococcal capsule was a required signal for aggregate formation. Macrophage aggregation coincided with NFκB activity, and the formation of these aggregates is mediated by leukotriene B4 (LTB4) produced by neutrophils. Depletion, inhibition, or genetic deletion of leukotriene A4 hydrolase (Lta4h), which catalyzes the last step in LTB4 synthesis, resulted in the absence of macrophage aggregation. Larvae with impaired neutrophil function also had impaired macrophage aggregation; however, aggregate formation was partially rescued with the addition of exogenous LTB4. Neutrophil-specific expression of lta4h was sufficient to rescue macrophage aggregation in Lta4h-deficient larvae and increased host survival following infection. In summary, our findings highlight a novel innate immune response to infection in which specific bacterial products drive neutrophils that modulate macrophage behavior through eicosanoid signaling.

Cell motility is required for diverse biological processes including development, homing of immune cells, wound healing, and cancer cell invasion. Motile neutrophils exhibit a polarized morphology characterized by the formation of leading-edge pseudopods and a highly contractile cell rear known as the uropod. Although it is known that perturbing uropod formation impairs neutrophil migration, the role of the uropod in cell polarization and motility remains incompletely understood. Here we discuss cell intrinsic mechanisms that regulate neutrophil polarization and motility, with a focus on the uropod, and examine how relationships among regulatory mechanisms change when cells change their direction of migration.

Obstacles to bacterial survival and replication in the cytosol of host cells, and the mechanisms used by bacterial pathogens to adapt to this niche are not well understood. Listeria monocytogenes is a well-studied Gram-positive foodborne pathogen that has evolved to invade and replicate within the host cell cytosol; yet the mechanisms by which it senses and responds to stress to survive in the cytosol are largely unknown. To assess the role of the L. monocytogenes penicillin-binding-protein and serine/threonine associated (PASTA) kinase PrkA in stress responses, cytosolic survival and virulence, we constructed a ΔprkA deletion mutant. PrkA was required for resistance to cell wall stress, growth on cytosolic carbon sources, intracellular replication, cytosolic survival, inflammasome avoidance and ultimately virulence in a murine model of Listeriosis. In Bacillus subtilis and Mycobacterium tuberculosis, homologues of PrkA phosphorylate a highly conserved protein of unknown function, YvcK. We found that, similar to PrkA, YvcK is also required for cell wall stress responses, metabolism of glycerol, cytosolic survival, inflammasome avoidance and virulence. We further demonstrate that similar to other organisms, YvcK is directly phosphorylated by PrkA, although the specific site(s) of phosphorylation are not highly conserved. Finally, analysis of phosphoablative and phosphomimetic mutants of YvcK in vitro and in vivo demonstrate that while phosphorylation of YvcK is irrelevant to metabolism and cell wall stress responses, surprisingly, a phosphomimetic, nonreversible negative charge of YvcK is detrimental to cytosolic survival and virulence in vivo. Taken together our data identify two novel virulence factors essential for cytosolic survival and virulence of L. monocytogenes. Furthermore, our data demonstrate that regulation of YvcK phosphorylation is tightly controlled and is critical for virulence. Finally, our data suggest that yet to be identified substrates of PrkA are essential for cytosolic survival and virulence of L. monocytogenes and illustrate the importance of studying protein phosphorylation in the context of infection., Author Summary Infection with intracellular pathogens causes a majority of the global infectious disease associated mortality. A number of intracellular pathogens must directly access the host cytosol in order to cause disease; however, non-cytosol adapted bacteria do not survive or replicate upon access to the cytosol. The mechanisms cytosolic pathogens use to adapt to this niche are largely unknown. The model cytosolic bacterial pathogen Listeria monocytogenes contains a single penicillin-binding-protein and serine/threonine associated (PASTA) kinase, PrkA. In other bacteria, PASTA kinases bind cell wall fragments and phosphorylate downstream effectors involved in cell wall synthesis, central metabolism, virulence, cell division, and biofilm formation. We demonstrate that in L. monocytogenes, PrkA is required for cell wall homeostasis, growth under nutrient limiting conditions, survival and replication in host cells, and virulence in vivo. Furthermore, we identify a highly conserved protein of unknown function, YvcK, as a PrkA substrate. We demonstrate that L. monocytogenes YvcK is similarly required for cell wall stress responses, growth on glycerol, cytosolic survival and virulence in vivo. Surprisingly, a phosphomimetic, nonreversible negative charge at the phosphorylation sites on YvcK inactivates functions of the protein related to intracellular survival and virulence, suggesting that the identification of PASTA kinase substrates phosphorylated during infection will be critical to our understanding of this central regulator metabolism, cell wall homeostasis and ultimately virulence.

While β-lactam antibiotics are a critical part of the antimicrobial arsenal, they are frequently compromised by various resistance mechanisms, including changes in penicillin binding proteins of the bacterial cell wall. Genetic deletion of the p enicillin binding protein a nd s erine/ t hreonine kinase- a ssociated protein (PASTA) kinase in methicillin-resistant Staphylococcus aureus (MRSA) has been shown to restore β-lactam susceptibility. However, the mechanism remains unclear, and whether pharmacologic inhibition would have the same effect is unknown. In this study, we found that deletion or pharmacologic inhibition of the PASTA kinase in Listeria monocytogenes by the nonselective kinase inhibitor staurosporine results in enhanced susceptibility to both aminopenicillin and cephalosporin antibiotics. Resistance to vancomycin, another class of cell wall synthesis inhibitors, or antibiotics that inhibit protein synthesis was unaffected by staurosporine treatment. Phosphorylation assays with purified kinases revealed that staurosporine selectively inhibited the PASTA kinase of L. monocytogenes (PrkA). Importantly, staurosporine did not inhibit a L. monocytogenes kinase without a PASTA domain (Lmo0618) or the PASTA kinase from MRSA (Stk1). Finally, inhibition of PrkA with a more selective kinase inhibitor, AZD5438, similarly led to sensitization of L. monocytogenes to β-lactam antibiotics. Overall, these results suggest that pharmacologic targeting of PASTA kinases can increase the efficacy of β-lactam antibiotics.

Corneal epithelial homeostasis and regeneration are sustained by limbal stem cells (LSCs)1–3, and LSC deficiency is a major cause of blindness worldwide4. Transplantation is often the only therapeutic option available to patients with LSC deficiency. However, while transplant success depends foremost on LSC frequency within grafts5, a gene allowing for prospective LSC enrichment has not been identified so far5. Here we show that ATP-binding cassette, sub-family B, member 5 (ABCB5)6,7 marks LSCs and is required for LSC maintenance, corneal development and repair. Furthermore, we demonstrate that prospectively isolated human or murine ABCB5-positive LSCs possess the exclusive capacity to fully restore the cornea upon grafting to LSC-deficient mice in xenogeneic or syngeneic transplantation models. ABCB5 is preferentially expressed on label-retaining LSCs2 in mice and p63α-positive LSCs8 in humans. Consistent with these findings, ABCB5-positive LSC frequency is reduced in LSC-deficient patients. Abcb5 loss of function in Abcb5 knockout mice causes depletion of quiescent LSCs due to enhanced proliferation and apoptosis, and results in defective corneal differentiation and wound healing. Our results from gene knockout studies, LSC tracing and transplantation models, as well as phenotypic and functional analyses of human biopsy specimens, provide converging lines of evidence that ABCB5 identifies mammalian LSCs. Identification and prospective isolation of molecularly defined LSCs with essential functions in corneal development and repair has important implications for the treatment of corneal disease, particularly corneal blindness due to LSC deficiency.