The Francisella Tularensis Proteome and its Recognition by Antibodies

Francisella tularensis is the causative agent of a spectrum of diseases collectively known as tularemia. The extreme virulence of the pathogen in humans, combined with the low infectious dose and the ease of dissemination by aerosol have led to concerns about its abuse as a bioweapon. Until recently, nothing was known about the virulence mechanisms and even now, there is still a relatively poor understanding of pathogen virulence. Completion of increasing numbers of Francisella genome sequences, combined with comparative genomics and proteomics studies, are contributing to the knowledge in this area. Tularemia may be treated with antibiotics, but there is currently no licensed vaccine. An attenuated strain, the Live Vaccine Strain (LVS) has been used to vaccinate military and at risk laboratory personnel, but safety concerns mean that it is unlikely to be licensed by the FDA for general use. Little is known about the protective immunity induced by vaccination with LVS, in humans or animal models. Immunoproteomics studies with sera from infected humans or vaccinated mouse strains, are being used in gel-based or proteome microarray approaches to give insight into the humoral immune response. In addition, these data have the potential to be exploited in the identification of new diagnostic or protective antigens, the design of next generation live vaccine strains, and the development of subunit vaccines. Herein, we briefly review the current knowledge from Francisella comparative proteomics studies and then focus upon the findings from immunoproteomics approaches

Tularaemia: A challenging zoonosis

In recent years, several emerging zoonotic vector-borne infections with potential impact on human health have been identified in Europe, including tularaemia, caused by Francisella tularensis.This remarkable pathogen, one of the most virulent microorganisms currently known, has been detected in increasingly new settings and in a wide range of wild species, including lagomorphs, rodents, carnivores, fish and invertebrate arthropods. Also, a renewed concern has arisen with regard to F. tularensis: its potential use by bioterrorists. Based on the information published concerning the latest outbreaks, the aim of this paper is to review the main features of the agent, its biology, immunology and epidemiology. Moreover, special focus will be given to zoonotic aspects of the disease, as tularaemia outbreaks in human populations have been frequently associated with disease in animals

<i>Pseudomonas aeruginosa</i> type IV minor pilins and PilY1 regulate virulence by modulating FimS-AlgR activity

<div><p>Type IV pili are expressed by a wide range of prokaryotes, including the opportunistic pathogen <i>Pseudomonas aeruginosa</i>. These flexible fibres mediate twitching motility, biofilm maturation, surface adhesion, and virulence. The pilus is composed mainly of major pilin subunits while the low abundance minor pilins FimU-PilVWXE and the putative adhesin PilY1 prime pilus assembly and are proposed to form the pilus tip. The minor pilins and PilY1 are encoded in an operon that is positively regulated by the FimS-AlgR two-component system. Independent of pilus assembly, PilY1 was proposed to be a mechanosensory component that—in conjunction with minor pilins—triggers up-regulation of acute virulence phenotypes upon surface attachment. Here, we investigated the link between the minor pilins/PilY1 and virulence. <i>pilW</i>, <i>pilX</i>, and <i>pilY1</i> mutants had reduced virulence towards <i>Caenorhabditis elegans</i> relative to wild type or a major pilin mutant, implying a role in pathogenicity that is independent of pilus assembly. We hypothesized that loss of specific minor pilins relieves feedback inhibition on FimS-AlgR, increasing transcription of the AlgR regulon and delaying <i>C</i>. <i>elegans</i> killing. Reporter assays confirmed that FimS-AlgR were required for increased expression of the minor pilin operon upon loss of select minor pilins. Overexpression of AlgR or its hyperactivation via a phosphomimetic mutation reduced virulence, and the virulence defects of <i>pilW</i>, <i>pilX</i>, and <i>pilY1</i> mutants required FimS-AlgR expression and activation. We propose that PilY1 and the minor pilins inhibit their own expression, and that loss of these proteins leads to FimS-mediated activation of AlgR that suppresses expression of acute-phase virulence factors and delays killing. This mechanism could contribute to adaptation of <i>P</i>. <i>aeruginosa</i> in chronic lung infections, as mutations in the minor pilin operon result in the loss of piliation and increased expression of AlgR-dependent virulence factors–such as alginate–that are characteristic of such infections.</p></div

AlgR hyperactivation delays killing.

<p>SK assays for (A) PA14 and (B) PAO1 <i>fimS</i>, <i>algR</i>, <i>algR</i>_D54A, and <i>algR</i>_D54E mutants. The <i>fimS</i>, <i>algR</i>, and <i>algR</i>_D54A mutants had WT virulence, while the <i>algR</i>_D54E mutants showed delays in killing. For (A) and (B), asterisks indicate strains that were significantly different from WT by Gehan-Breslow-Wilcoxon test at p = 0.05 (p = 0.01 with a Bonferroni correction), n = 3 trials.</p

Model for regulation of the MPs and virulence by FimS-AlgR.

<p>(A) Loss or inactivation of FimS-AlgR results in sustained WT (acute) virulence towards <i>C</i>. <i>elegans</i>. Under normal conditions, PilVWXY1 suppress FimS activation of AlgR, leading to reduced expression of the MPs and increased expression of acute virulence factors. These phenotypes are mimicked by genetic inactivation of AlgR (D54A) or deletion of <i>fimS</i> or <i>algR</i>. (B) Loss of PilVWXY1 frees FimS to activate AlgR, leading to increased expression of the MPs, reduced expression of acute virulence factors, and delayed nematode killing. Hyperactivating mutations in AlgR (D54E) phenocopy this mechanism. Abbreviations: <i>fimU</i>, U (magenta); <i>pilV</i>, V (orange); <i>pilW</i>, W (teal); <i>pilX</i>, X (pink); <i>pilY1</i>, Y1 (purple); <i>pilE</i>, E (green). Yellow star indicates phosphorylation.</p

PilWXY1 contribute to T4P-independent virulence.

<p>(A) SK assays for PA14 <i>pilA</i>, <i>fimU</i>, <i>pilV</i>, <i>pilW</i>, <i>pilX</i>, <i>pilY1</i>, and <i>pilE</i> mutants. Synchronized L4 worms were seeded onto SK plates and scored for death every 24 h, then plotted as “percent survival” over the course of the assay. “Day” represents the number of days after L4 on which the plates were scored. PA14 <i>fimU</i> and <i>pilE</i> mutants had similar virulence to WT, <i>pilA</i> and <i>pilV</i> mutants were slightly less virulent than WT, and <i>pilW</i>, <i>pilX</i>, and <i>pilY1</i> mutants killed more slowly than all other strains tested. (B) SK assays for PAO1 <i>pilA</i>, <i>fimU</i>, <i>pilV</i>, <i>pilW</i>, <i>pilX</i>, <i>pilY1</i>, and <i>pilE</i> mutants. The PAO1 <i>pilE</i> mutant had similar virulence to WT, the <i>pilA</i> mutant was slightly less virulent, and <i>fimU</i>, <i>pilV</i>, <i>pilW</i>, <i>pilX</i>, and <i>pilY1</i> mutants showed significant delays in killing. In (A) and (B), asterisks indicate strains that were significantly different from a <i>pilA</i> mutant by Gehan-Breslow-Wilcoxon test at p = 0.05 (p = 0.00625 with a Bonferroni correction), n = 3 trials.</p