Na/H exchanger NHE1 acts upstream of rho GTPases to promote neurite outgrowth.

Na+/H+ exchanger NHE1, a major determinant of intracellular pH (pHi) in mammalian central neurons, promotes neurite outgrowth under both basal and netrin-1-stimulated conditions. The small GTP binding proteins and their effectors have a dominant role in netrin-1-stimulated neurite outgrowth. Since NHE1 has been shown previously to work downstream of the Rho GTPases-mediated polarized membrane protrusion in non-neuronal cells, we examined whether NHE1 has a similar relationship with Cdc42, Rac1 and RhoA in neuronal morphogenesis. Interestingly, our results suggest the possibility that NHE1 acting upstream of Rho GTPases to promote neurite outgrowth induced by netrin-1. First, we found that netrin-1-induced increases in the activities of Rho GTPases using FRET (Forster Resonance Energy Transfer) analyses in individual growth cones; furthermore, their increased activities were abolished by cariporide, a specific NHE1 inhibitor. Second, NHE1 inhibition had no effect on neurite retraction induced by L-α-Lysophosphatidic acid (LPA), a potent RhoA activator. The regulation of Rho GTPases by NHE1 was further evidenced by reduced Rac1, Cdc42 and RhoA activities in NHE1-null neurons. Taken together, our findings suggest that NHE1-dependent neuronal morphogenesis involves the activation of Rho-family of small GTPases.

Defects in phosphate acquisition and storage influence virulence of Cryptococcus neoformans.

Nutrient acquisition and sensing are critical aspects of microbial pathogenesis. Previous transcriptional profiling indicated that the fungal pathogen Cryptococcus neoformans, which causes meningoencephalitis in immunocompromised individuals, encounters phosphate limitation during proliferation in phagocytic cells. We therefore tested the hypothesis that phosphate acquisition and polyphosphate metabolism are important for cryptococcal virulence. Deletion of the high-affinity uptake system interfered with growth on low-phosphate medium, perturbed the formation of virulence factors (capsule and melanin), reduced survival in macrophages, and attenuated virulence in a mouse model of cryptococcosis. Additionally, analysis of nutrient sensing functions for C. neoformans revealed regulatory connections between phosphate acquisition and storage and the iron regulator Cir1, cyclic AMP (cAMP)-dependent protein kinase A (PKA), and the calcium-calmodulin-activated protein phosphatase calcineurin. Deletion of the VTC4 gene encoding a polyphosphate polymerase blocked the ability of C. neoformans to produce polyphosphate. The vtc4 mutant behaved like the wild-type strain in interactions with macrophages and in the mouse infection model. However, the fungal load in the lungs was significantly increased in mice infected with vtc4 deletion mutants. In addition, the mutant was impaired in the ability to trigger blood coagulation in vitro, a trait associated with polyphosphate. Overall, this study reveals that phosphate uptake in C. neoformans is critical for virulence and that its regulation is integrated with key signaling pathways for nutrient sensing.

Salmonella SPI-1-mediated neutrophil recruitment during enteric colitis is associated with reduction and alteration in intestinal microbiota.

Gastrointestinal infections involve an interactive tripartite relationship between the invading pathogen, the host, and the host's resident intestinal microbiota. To characterize the host inflammatory response and microbiota alterations during enteric salmonellosis, C57BL/6 mice were pre-treated with a low dose of streptomycin (LD model) and then infected with S. typhimurium strains, including mutants in the two Type III secretion systems, SPI-1 and SPI-2 (invAmut and ssaRmut, respectively). Cecal colonization and inflammation in the LD model were evaluated to assess infection success and progression, and compared to the traditional high dose (HD) model. Perturbations to the microbial community in the LD model were assessed via evaluation of total microbial numbers, the proportion of intestinal γ-Proteobacteria and tRFLP analysis. In the LD model, consistently high colonization by the parental strain (WT) and invAmut S. typhimurium was associated with significant intestinal pathology. However, microbial community profiles were more similar both in numbers and composition between mice infected with the mutant strains, than with the WT strain. Consequently, significant infection-induced inflammation did not always produce similar microbiota perturbations. Large numbers of luminal neutrophils were observed in the ceca of WT-infected, but not in invAmut or ssaRmut infected mice. Neutrophils were thus implicated as a potential mediator of microbiota perturbations during WT enteric salmonellosis. These studies offer a new model of S. typhimurium-induced intestinal disease that retains the three participants of the disease process and further defines the role of virulence factors, the host microbiota, and inflammation in S. typhimurium-induced intestinal disease.

Antibiotic-induced perturbations of the intestinal microbiota alter host susceptibility to enteric infection.

Intestinal microbiota comprises microbial communities that reside in the gastrointestinal tract and are critical to normal host physiology. Understanding the microbiota's role in host response to invading pathogens will further advance our knowledge of host-microbe interactions. Salmonella enterica serovar Typhimurium was used as a model enteric pathogen to investigate the effect of intestinal microbiota perturbation on host susceptibility to infection. Antibiotics were used to perturb the intestinal microbiota. C57BL/6 mice were treated with clinically relevant doses of streptomycin and vancomycin in drinking water for 2 days, followed by oral infection with Salmonella enterica serovar Typhimurium. Alterations in microbiota composition and numbers were evaluated by fluorescent in situ hybridization, differential plating, and Sybr green staining. Antibiotics had a dose-dependent effect on intestinal microbiota composition. The chosen antibiotic regimen did not significantly alter the total numbers of intestinal bacteria but altered the microbiota composition. Greater preinfection perturbations in the microbiota resulted in increased mouse susceptibility to Salmonella serovar Typhimurium intestinal colonization, greater postinfection alterations in the microbiota, and more severe intestinal pathology. These results suggest that antibiotic treatment alters the balance of the microbial community, which predisposes the host to Salmonella serovar Typhimurium infection, demonstrating the importance of a healthy microbiota in host response to enteric pathogens.

Transcriptional regulation by protein kinase A in Cryptococcus neoformans.

A defect in the PKA1 gene encoding the catalytic subunit of cyclic adenosine 5'-monophosphate (cAMP)-dependent protein kinase A (PKA) is known to reduce capsule size and attenuate virulence in the fungal pathogen Cryptococcus neoformans. Conversely, loss of the PKA regulatory subunit encoded by pkr1 results in overproduction of capsule and hypervirulence. We compared the transcriptomes between the pka1 and pkr1 mutants and a wild-type strain, and found that PKA influences transcript levels for genes involved in cell wall synthesis, transport functions such as iron uptake, the tricarboxylic acid cycle, and glycolysis. Among the myriad of transcriptional changes in the mutants, we also identified differential expression of ribosomal protein genes, genes encoding stress and chaperone functions, and genes for secretory pathway components and phospholipid synthesis. The transcriptional influence of PKA on these functions was reminiscent of the linkage between transcription, endoplasmic reticulum stress, and the unfolded protein response in Saccharomyces cerevisiae. Functional analyses confirmed that the PKA mutants have a differential response to temperature stress, caffeine, and lithium, and that secretion inhibitors block capsule production. Importantly, we also found that lithium treatment limits capsule size, thus reinforcing potential connections between this virulence trait and inositol and phospholipid metabolism. In addition, deletion of a PKA-regulated gene, OVA1, revealed an epistatic relationship with pka1 in the control of capsule size and melanin formation. OVA1 encodes a putative phosphatidylethanolamine-binding protein that appears to negatively influence capsule production and melanin accumulation. Overall, these findings support a role for PKA in regulating the delivery of virulence factors such as the capsular polysaccharide to the cell surface and serve to highlight the importance of secretion and phospholipid metabolism as potential targets for anti-cryptococcal therapy.