From insect to man: Photorhabdus sheds light on the emergence of human pathogenicity

Photorhabdus are highly effective insect pathogenic bacteria that exist in a mutualistic relationship with Heterorhabditid nematodes. Unlike other members of the genus, Photorhabdus asymbiotica can also infect humans. Most Photorhabdus cannot replicate above 34°C, limiting their host-range to poikilothermic invertebrates. In contrast, P. asymbiotica must necessarily be able to replicate at 37°C or above. Many well-studied mammalian pathogens use the elevated temperature of their host as a signal to regulate the necessary changes in gene expression required for infection. Here we use RNA-seq, proteomics and phenotype microarrays to examine temperature dependent differences in transcription, translation and phenotype of P. asymbiotica at 28°C versus 37°C, relevant to the insect or human hosts respectively. Our findings reveal relatively few temperature dependant differences in gene expression. There is however a striking difference in metabolism at 37°C, with a significant reduction in the range of carbon and nitrogen sources that otherwise support respiration at 28°C. We propose that the key adaptation that enables P. asymbiotica to infect humans is to aggressively acquire amino acids, peptides and other nutrients from the human host, employing a so called “nutritional virulence” strategy. This would simultaneously cripple the host immune response while providing nutrients sufficient for reproduction. This might explain the severity of ulcerated lesions observed in clinical cases of Photorhabdosis. Furthermore, while P. asymbiotica can invade mammalian cells they must also resist immediate killing by humoral immunity components in serum. We observed an increase in the production of the insect Phenol-oxidase inhibitor Rhabduscin normally deployed to inhibit the melanisation immune cascade. Crucially we demonstrated this molecule also facilitates protection against killing by the alternative human complement pathway

Mycobacterium tuberculosis Exploits Asparagine to Assimilate Nitrogen and Resist Acid Stress during Infection

Mycobacterium tuberculosis is an intracellular pathogen. Within macrophages, M. tuberculosis thrives in a specialized membrane-bound vacuole, the phagosome, whose pH is slightly acidic, and where access to nutrients is limited. Understanding how the bacillus extracts and incorporates nutrients from its host may help develop novel strategies to combat tuberculosis. Here we show that M. tuberculosis employs the asparagine transporter AnsP2 and the secreted asparaginase AnsA to assimilate nitrogen and resist acid stress through asparagine hydrolysis and ammonia release. While the role of AnsP2 is partially spared by yet to be identified transporter(s), that of AnsA is crucial in both phagosome acidification arrest and intracellular replication, as an M. tuberculosis mutant lacking this asparaginase is ultimately attenuated in macrophages and in mice. Our study provides yet another example of the intimate link between physiology and virulence in the tubercle bacillus, and identifies a novel pathway to be targeted for therapeutic purposes. © 2014 Gouzy et al

Leishmania infantum Asparagine Synthetase A Is Dispensable for Parasites Survival and Infectivity

A growing interest in asparagine (Asn) metabolism has currently been observed in cancer and infection fields. Asparagine synthetase (AS) is responsible for the conversion of aspartate into Asn in an ATP-dependent manner, using ammonia or glutamine as a nitrogen source. There are two structurally distinct AS: the strictly ammonia dependent, type A, and the type B, which preferably uses glutamine. Absent in humans and present in trypanosomatids, AS-A was worthy of exploring as a potential drug target candidate. Appealingly, it was reported that AS-A was essential in Leishmania donovani, making it a promising drug target. In the work herein we demonstrate that Leishmania infantum AS-A, similarly to Trypanosoma spp. and L. donovani, is able to use both ammonia and glutamine as nitrogen donors. Moreover, we have successfully generated LiASA null mutants by targeted gene replacement in L. infantum, and these parasites do not display any significant growth or infectivity defect. Indeed, a severe impairment of in vitro growth was only observed when null mutants were cultured in asparagine limiting conditions. Altogether our results demonstrate that despite being important under asparagine limitation, LiAS-A is not essential for parasite survival, growth or infectivity in normal in vitro and in vivo conditions. Therefore we exclude AS-A as a suitable drug target against L. infantum parasites

Human Epithelial Cells Discriminate between Commensal and Pathogenic Interactions with <i>Candida albicans</i>

<div><p>The commensal fungus, <i>Candida albicans</i>, can cause life-threatening infections in at risk individuals. <i>C</i>. <i>albicans</i> colonizes mucosal surfaces of most people, adhering to and interacting with epithelial cells. At low concentrations, <i>C</i>. <i>albicans</i> is not pathogenic nor does it cause epithelial cell damage <i>in vitro</i>; at high concentrations, <i>C</i>. <i>albicans</i> causes mucosal infections and kills epithelial cells <i>in vitro</i>. Here we show that while there are quantitative dose-dependent differences in exposed epithelial cell populations, these reflect a fundamental qualitative difference in host cell response to <i>C</i>. <i>albicans</i>. Using transcriptional profiling experiments and real time PCR, we found that wild-type <i>C</i>. <i>albicans</i> induce dose-dependent responses from a FaDu epithelial cell line. However, real time PCR and Western blot analysis using a high dose of various <i>C</i>. <i>albicans</i> strains demonstrated that these dose-dependent responses are associated with ability to promote host cell damage. Our studies support the idea that epithelial cells play a key role in the immune system by monitoring the microbial community at mucosal surfaces and initiating defensive responses when this community is dysfunctional. This places epithelial cells at a pivotal position in the interaction with <i>C</i>. <i>albicans</i> as epithelial cells themselves promote <i>C</i>. <i>albicans</i> stimulated damage.</p></div

Effect of <i>C</i>. <i>albicans</i> mutations on host transcriptional responses.

<p>RT-PCR for IL-8, SERPIN E1 (SER E1), IL-6, IL1⍺, DUSP1, IL-24, and DUSP6 transcription, expressed in relative ng (y-axis). cDNA derived from mRNA purified from FaDu epithelial cell monolayers infected with wild-type <i>C</i>. <i>albicans</i> (■), <i>rim101Δ/Δ</i> (▲), mutant cells, <i>efg1 cph1Δ/Δ</i> (×) mutant cells, <i>S</i>. <i>cerevisiae</i> (*), or non-infected (◆) at defined time points (x-axis). A single time course experiment is shown, but analogous results were obtained in two replicate experiments.</p

Common Epithelial Cell Responses to High and Low Dose <i>C</i>. <i>albicans</i>.

<p>Common Epithelial Cell Responses to High and Low Dose <i>C</i>. <i>albicans</i>.</p

Relevant GO categories of Induced Genes.

<p>Relevant GO categories of Induced Genes.</p

Primer sets used for RT-PCR.

<p>Primer sets used for RT-PCR.</p

Inflammatory Response Genes.

<p>Inflammatory Response Genes.</p