Type III secretion systems (T3SSs) are used by Gram-negative pathogens to form pores in host membranes and deliver virulence-associated effector proteins inside host cells. In pathogenic Yersinia, the T3SS pore-forming proteins are YopB and YopD. Mammalian cells recognize the Yersinia T3SS, leading to a host response that includes secretion of the inflammatory cytokine interleukin-1␤ (IL-1␤), Toll-like receptor (TLR)-independent expression of the stress-associated transcription factor Egr1 and the inflammatory cytokine tumor necrosis factor alpha (TNF-␣), and host cell death. The known Yersinia T3SS effector proteins are dispensable for eliciting these responses, but YopB is essential. Three models describe how the Yersinia T3SS might trigger inflammation: (i) mammalian cells sense YopBD-mediated pore formation, (ii) innate immune stimuli gain access to the host cytoplasm through the YopBD pore, and/or (iii) the YopB-YopD translocon itself or its membrane insertion is proinflammatory. To test these models, we constructed a Yersinia pseudotuberculosis mutant expressing YopD devoid of its predicted transmembrane domain (YopD ⌬TM ) and lacking the T3SS cargo proteins YopHE-MOJTN. This mutant formed pores in macrophages, but it could not mediate translocation of effector proteins inside host cells. Importantly, this mutant did not elicit rapid host cell death, IL-1␤ secretion, or TLR-independent Egr1 and TNF-␣ expression. These data suggest that YopBD-mediated translocation of unknown T3SS cargo leads to activation of host pathways influencing inflammation, cell death, and response to stress. As the YopD ⌬TM Y. pseudotuberculosis mutant formed somewhat smaller pores with delayed kinetics, an alternative model is that the wild-type YopB-YopD translocon is specifically sensed by host cells.

Streptococcus pneumoniae is a major cause of pneumonia, wherein infection of respiratory mucosa drives a robust influx of neutrophils. We have previously shown that S. pneumoniae infection of the respiratory epithelium induces the production of the 12-lipoxygenase (12-LOX)-dependent lipid inflammatory mediator hepoxilin A3, which promotes recruitment of neutrophils into the airways, tissue damage, and lethal septicemia. Pneumolysin (PLY), a member of the cholesterol-dependent cytolysin (CDC) family, is a major S. pneumoniae virulence factor that generates ∼25-nm diameter pores in eukaryotic membranes and promotes acute inflammation, tissue damage, and bacteremia. We show that a PLY-deficient S. pneumoniae mutant was impaired in triggering human neutrophil transepithelial migration in vitro. Ectopic production of PLY endowed the nonpathogenic Bacillus subtilis with the ability to trigger neutrophil recruitment across human-cultured monolayers. Purified PLY, several other CDC family members, and the a-toxin of Clostridium septicum, which generates pores with cross-sectional areas nearly 300 times smaller than CDCs, reproduced this robust neutrophil transmigration. PLY non-pore-forming point mutants that are trapped at various stages of pore assembly did not recruit neutrophils. PLY triggered neutrophil recruitment in a 12-LOX-dependent manner in vitro. Instillation of wild-type PLY but not inactive derivatives into the lungs of mice induced robust 12-LOXdependent neutrophil migration into the airways, although residual inflammation induced by PLY in 12-LOX-deficient mice indicates that 12-LOX-independent pathways also contribute to PLY-triggered pulmonary inflammation. These data indicate that PLY is an important factor in promoting hepoxilin A3-dependent neutrophil recruitment across pulmonary epithelium in a pore-dependent fashion.

Neutrophil transepithelial migration is a fundamental process that facilitates the rapid trafficking of neutrophils to inflammatory foci and occurs across a diverse range of tissues. For decades there has been widespread interest in understanding the mechanisms that drive this migratory process in response to different pathogens and organ systems. This has led to the successful integration of key findings on neutrophil transepithelial migration from the intestines, lungs, liver, genitourinary tract, and other tissues into a single, cohesive model. However, recent studies have identified organ specific differences in neutrophil transepithelial migration. These findings support a model where the tissue in concert with the pro-inflammatory stimuli dictate a unique collection of signals that drive neutrophil trafficking. This review focuses on the mechanisms that drive neutrophil transepithelial migration in response to microbial infection of a single organ, the lung. Herein we provide a detailed analysis of the adhesion molecules and chemoattractants that contribute to the recruitment of neutrophil into the airways. We also highlight important advances in experimental models for studying neutrophil transepithelial migration in the lung over the last decade.

Cryptosporidium is a leading cause of diarrhea and death in young children and untreated AIDS patients and causes waterborne outbreaks. Pathogenic mechanisms underlying diarrhea and intestinal dysfunction are poorly understood.