"Francisella tularensis subsp. novicida" intranasal infection causes a rapid pneumonia in mice with mortality at 4 to 6 days with a low dose of bacteria (10 2 bacteria). The short time to death suggests that there is a failure of the innate immune response. As the neutrophil is often the first cell type to infiltrate sites of infection, we focused on the emigration of neutrophils in this infection, as well as cytokines involved in their recruitment. The results indicated that there was a significant delay in the influx of neutrophils into the bronchoalveolar lavage fluid of F. tularensis subsp. novicida-infected mice. The delay in neutrophil recruitment in F. tularensis subsp. novicida-infected mice correlated with a delay in the upregulation of multiple proinflammatory cytokines and chemokines, as well as a delay in caspase-1 activation. Strikingly, the initial delay in the upregulation of cytokines through 1 day postinfection was followed by profound upregulation of multiple cytokines and chemokines to levels consistent with hypercytokinemia described for severe sepsis. This finding was further supported by a bacteremia and the cellular relocalization and release of high-mobility group box-1 and S100A9, both of which are damage-associated molecular pattern molecules and are known to be mediators of severe sepsis.

The virulence mechanisms of Francisella tularensis, the causative agent of severe pneumonia in humans and a CDC category A bioterrorism agent, are not fully defined. As sepsis is the leading cause of mortality associated with respiratory infections, we determined whether, in the absence of any known bacterial toxins, a deregulated host response resulting in sepsis syndrome is associated with lethality of respiratory infection with the virulent human Type A strain SchuS4 of F. tularensis. The C57BL/6 mice infected intranasally with a lethal dose of SchuS4 exhibited high bacterial burden in systemic organs and blood indicative of bacteremia. In correlation, infected mice displayed severe tissue pathology and associated cell death in lungs, liver and spleen. Consistent with our studies with murine model strain F. novicida, infection with SchuS4 caused an initial delay in upregulation of inflammatory mediators followed by development of severe sepsis characterized by exaggerated cytokine release, upregulation of cardiovascular injury markers and sepsis mediator alarmins S100A9 and HMGB-1. This study shows that pulmonary tularemia caused by the Type A strain of F. tularensis results in a deregulated host response leading to severe sepsis and likely represents the major cause of mortality associated with this virulent pathogen.

Serratia marcescens, a member of the carbapenem-resistant Enterobacteriaceae, is an important emerging pathogen that causes a wide variety of nosocomial infections, spreads rapidly within hospitals, and has a systemic mortality rate of <41%. Despite multiple clinical descriptions of S. marcescens nosocomial pneumonia, little is known regarding the mechanisms of bacterial pathogenesis and the host immune response. To address this gap, we developed an oropharyngeal aspiration model of lethal and sublethal S. marcescens pneumonia in BALB/c mice and extensively characterized the latter. Lethal challenge (>4.0 ؋ 10 6 CFU) was characterized by fulminate hemorrhagic pneumonia with rapid loss of lung function and death. Mice challenged with a sublethal dose (<2.0 ؋ 10 6 CFU) rapidly lost weight, had diminished lung compliance, experienced lung hemorrhage, and responded to the infection with extensive neutrophil infiltration and histopathological changes in tissue architecture. Neutrophil extracellular trap formation and the expression of inflammatory cytokines occurred early after infection. Mice depleted of neutrophils were exquisitely susceptible to an otherwise nonlethal inoculum, thereby demonstrating the requirement for neutrophils in host protection. Mutation of the genes encoding the cytolysin ShlA and its transporter ShlB resulted in attenuated S. marcescens strains that failed to cause profound weight loss, extended illness, hemorrhage, and prolonged lung pathology in mice. This study describes a model of S. marcescens pneumonia that mimics known clinical features of human illness, identifies neutrophils and the toxin ShlA as a key factors important for defense and infection, respectively, and provides a solid foundation for future studies of novel therapeutics for this important opportunistic pathogen. Serratia marcescens is a facultative anaerobic, rod-shaped, Gram-negative bacterium that is ubiquitous in water, in soil, and on plant surfaces. S. marcescens is also an antibiotic-resistant opportunistic pathogen and is among the top 10 causative agents of bloodstream bacterial infections in North America, with a mortality rate of 41% (1-3). In newborns and immunocompromised and intensive care patients, S. marcescens can cause severe infections such as pneumonia (4), bloodstream infections (5), and urinary tract infections, surgical site infections, and ocular infections (6). S. marcescens infections are most often associated with the hospital environment (5), but community-acquired infections are now increasingly diagnosed (7).S. marcescens has acquired notoriety in the last 20 years because of its resistance to multiple antibiotics (7,8). An 8-year surveillance study in Taiwan identified multiple S. marcescens strains that were resistant to the antibiotics ciprofloxacin and levofloxacin (9). Multiple studies have revealed an alarming increase in S. marcescens resistance to carbapenem and other ␤-lactam antibiotics (8, 10, 11). As such, S. marcescens is considered a member of the carbapenem-resistant Enterobac...

The macrophage is a versatile cell type that can sense and respond to a particular need based on the conditions of the microenvironment. Some studies have recently suggested that pathogens can directly influence the polarization of macrophages. As Francisella infections are characterized by intense necrotic infiltrates in the lung as well as in distal sites of infection, we sought to investigate whether pulmonary Francisella infections could cause the polarization of alternatively activated macrophages (M2/aaMs). Our results indicate that Francisella infections can cause the polarization of M2/aaM in vivo and that macrophages can be polarized toward an M2/aaM phenotype more potently if dead cell debris is used for stimulation in the presence and absence of Francisella infections. Finally, we also demonstrate that efferocytosis is inhibited in macrophages infected with Francisella, thus providing a potential explanation for the lack of clearance and eventual accumulation of dead cell debris associated with this disease. Keywords: alternatively activated macrophages; tularemia; efferocytosis Macrophage activation and differentiation is currently an area of intense research. There are now several types of macrophages and at least two classification systems for macrophages. They include classically activated macrophages and alternatively activated macrophages (aaMs or M2). M2/aaMs can also be further classified into regulatory or wound-healing macrophages according to the markers they express as well as their functionality. 1,2 The unique subsets of M2/aaMs and their corresponding functions arise in response to a distinctive set of stimuli that have recently been shown to be either dependent or independent of interleukin-4. Interestingly, macrophages have also recently been proposed to be highly adaptable to the stimuli present in a given local milieu and can alter their activity to meet the needs present in a specific circumstance. 3 Tularemia is a disease caused by the Gram-negative coccobacillus species in the genus Francisella. The pneumonic form of the disease is characterized by an initial delay in the immune response of approximately 48-72 h. Following this delay, there is a massive influx of inflammatory cells 4,5 as well as the onset of hypercytokinemia and sepsis. 6,7 We have also recently observed that the fatal outcome of the disease correlates with the release of damage-associated molecular patterns (DAMPs) or alarmins that have been described to be potent inflammatory mediators derived from various modes of cell death. [8][9][10] Importantly, recent studies have also provided a link between endogenous immune mediators (DAMPs) released in cell death and their potential ability to affect macrophage polarization. [11][12][13] In light of these studies and in conjunction with the striking pathology associated with pulmonary Francisella infections, we hypothesized that the extent of cell death generated in response to infection could have an important role in macrophage polarization. The aim of this study w...

SummaryCryptococcus neoformans typically grows in a yeastlike morphology; however, under specific conditions the fungus can produce hyphae that are either dikaryotic or monokaryotic. In this study, we developed a simple method for inducing robust monokaryotic fruiting and combined the assay with Agrobacterium tumefaciens insertional mutagenesis to screen for hyphal mutants. A C. neoformans homologue of the Saccharomyces cerevisiae STE50 gene was identified and characterized. STE50 was found to be required for sexual reproduction and monokaryotic fruiting. Ste50p has conserved SAM and RA domains, as well as two SH3 domains specific to basidiomycetous Ste50 proteins. Analysis of protein-protein interaction showed that Ste50p can interact with Ste11p and Ste20p, and epistasis experiments placed STE50 between STE20 and STE11. Genetic analysis of the role of STE50 in sexual reproduction showed that it was required for all steps, from response to pheromone to production of hyphae. Analysis of the effect of individual Ste50p domains on sexual reproduction and monokaryotic fruiting revealed domain-specific effects for both processes. This study revealed that the C. neoformans STE50 gene has both conserved and novel functions during sexual reproduction and monokaryotic fruiting, and these functions are domain-dependent.