We determined that LVS and Schu S4 strains of the human pathogen Francisella tularensis express a siderophore when grown under iron-limiting conditions. We purified this siderophore by conventional column chromatography and high-pressure liquid chromatography and used mass spectrometric analysis to demonstrate that it is structurally similar to the polycarboxylate siderophore rhizoferrin. The siderophore promoted the growth of LVS and Schu S4 strains in iron-limiting media. We identified a potential siderophore biosynthetic gene cluster encoded by fslABCD in the F. tularensis genome. The first gene in the cluster, fslA, encodes a member of the superfamily of nonribosomal peptide synthetase-independent siderophore synthetases (NIS synthetases) characterized by the aerobactin synthetases IucA and IucC. We determined that fslA is transcribed as part of an operon with downstream gene fslB and that the expression of the locus is induced by iron starvation. A targeted in-frame nonpolar deletion of fslA in LVS resulted in the loss of siderophore expression and in a reduced ability of F. tularensis to grow under conditions of iron limitation. Siderophore activity and the ability to grow under iron limitation could be regained by introducing the fslA ؉ gene on a complementing plasmid. Our results suggest that the fslA-dependent siderophore is important for survival of F. tularensis in an iron-deficient environment.

The NLR gene family mediates host immunity to various acute pathogenic stimuli, but its role in chronic infection is not known. This paper addressed the role of NLRP3 (NALP3), its adaptor protein PYCARD (ASC), and caspase-1 during infection with Mycobacterium tuberculosis (Mtb). Mtb infection of macrophages in culture induced IL-1β secretion, and this requires the inflammasome components PYCARD, caspase-1, and NLRP3. However, in vivo Mtb aerosol infection of Nlrp3−/−, Casp-1−/−, and WT mice showed no differences in pulmonary IL-1β production, bacterial burden, or long-term survival. In contrast, a significant role was observed for Pycard in host protection during chronic Mtb infection, as shown by an abrupt decrease in survival of Pycard−/− mice. Decreased survival of Pycard−/− animals was associated with defective granuloma formation. These data demonstrate that PYCARD exerts a novel inflammasome-independent role during chronic Mtb infection by containing the bacteria in granulomas.

The ability of Mycobacterium tuberculosis to grow in macrophages is critical to the virulence of this important pathogen. One way M. tuberculosis is thought to maintain a hospitable niche in macrophages is by arresting the normal process of phagosomes maturing into acidified phagolysosomes. The process of phagosome maturation arrest by M. tuberculosis is not fully understood, and there has remained a need to firmly establish a requirement for phagosome maturation arrest for M. tuberculosis growth in macrophages. Other intracellular pathogens that control the phagosomal environment use specialized protein export systems to deliver effectors of phagosome trafficking to the host cell. In M. tuberculosis, the accessory SecA2 system is a specialized protein export system that is required for intracellular growth in macrophages. In studying the importance of the SecA2 system in macrophages, we discovered that SecA2 is required for phagosome maturation arrest. Shortly after infection, phagosomes containing a ⌬secA2 mutant of M. tuberculosis were more acidified and showed greater association with markers of late endosomes than phagosomes containing wild-type M. tuberculosis. We further showed that inhibitors of phagosome acidification rescued the intracellular growth defect of the ⌬secA2 mutant, which demonstrated that the phagosome maturation arrest defect of the ⌬secA2 mutant is responsible for the intracellular growth defect. This study demonstrates the importance of phagosome maturation arrest for M. tuberculosis growth in macrophages, and it suggests there are effectors of phagosome maturation that are exported into the host environment by the accessory SecA2 system.

Protein export is essential in all bacteria and many bacterial pathogens depend on specialized protein export systems for virulence. In Mycobacterium tuberculosis, the etiological agent of the disease tuberculosis, the conserved general secretion (Sec) and twin-arginine translocation (Tat) pathways perform the bulk of protein export and are both essential. M. tuberculosis also has specialized export pathways that transport specific subsets of proteins. One such pathway is the accessory SecA2 system, which is important for M. tuberculosis virulence. There are also specialized ESX export systems that function in virulence (ESX-1) or essential physiologic processes (ESX-3). The increasing prevalence of drug-resistant M. tuberculosis strains makes the development of novel drugs for tuberculosis an urgent priority. In this article, we discuss our current understanding of the protein export systems of M. tuberculosis and consider the potential of these pathways to be novel targets for tuberculosis drugs.

To subvert host defenses, Mycobacterium tuberculosis (Mtb) avoids being delivered to degradative phagolysosomes in macrophages by arresting the normal host process of phagosome maturation. Phagosome maturation arrest by Mtb involves multiple effectors and much remains unknown about this important aspect of Mtb pathogenesis. The SecA2 dependent protein export system is required for phagosome maturation arrest and consequently growth of Mtb in macrophages. To better understand the role of the SecA2 pathway in phagosome maturation arrest, we identified two effectors exported by SecA2 that contribute to this process: the phosphatase SapM and the kinase PknG. Then, utilizing the secA2 mutant of Mtb as a platform to study effector functions, we identified specific steps in phagosome maturation inhibited by SapM and/or PknG. By identifying a histidine residue that is essential for SapM phosphatase activity, we confirmed for the first time that the phosphatase activity of SapM is required for its effects on phagosome maturation in macrophages. We further demonstrated that SecA2 export of SapM and PknG contributes to the ability of Mtb to replicate in macrophages. Finally, we extended our understanding of the SecA2 pathway, SapM, and PknG by revealing that their contribution goes beyond preventing Mtb delivery to mature phagolysosomes and includes inhibiting Mtb delivery to autophagolysosomes. Together, our results revealed SapM and PknG to be two effectors exported by the SecA2 pathway of Mtb with distinct as well as cumulative effects on phagosome and autophagosome maturation. Our results further reveal that Mtb must have additional mechanisms of limiting acidification of the phagosome, beyond inhibiting recruitment of the V-ATPase proton pump to the phagosome, and they indicate differences between effects of Mtb on phagosome and autophagosome maturation.

The insulin-responsive 12-transmembrane transporter GLUT4 changes conformation between an inward-open state and an outward-open state to actively facilitate cellular glucose uptake. Because of the difficulties of generating conformational mAbs against complex and highly conserved membrane proteins, no reliable tools exist to measure GLUT4 at the cell surface, follow its trafficking, or detect the conformational state of the protein. Here we report the isolation and characterization of conformational mAbs that recognize the extracellular and intracellular domains of GLUT4, including mAbs that are specific for the inward-open and outward-open states of GLUT4. mAbs against GLUT4 were generated using virus-like particles to present this complex membrane protein in its native conformation and using a divergent host species (chicken) for immunization to overcome immune tolerance. As a result, the isolated mAbs recognize conformational epitopes on native GLUT4 in cells, with apparent affinities as high as 1 pM and with specificity for GLUT4 across the human membrane proteome. Epitope mapping using shotgun mutagenesis alanine scanning across the 509 amino acids of GLUT4 identified the binding epitopes for mAbs specific for the states of GLUT4 and allowed the comprehensive identification of the residues that functionally control the GLUT4 inward-open and outward-open states. The mAbs identified here will be valuable molecular tools for monitoring GLUT4 structure, function, and trafficking, for differentiating GLUT4 conformational states, and for the development of novel therapeutics for the treatment of diabetes.

The majority of nucleotide binding domain leucine rich repeats-containing (NLR) family members has yet to be functionally characterized. Of the described NLRs, most are considered to be proinflammatory and facilitate IL-1β production. However, a newly defined sub-group of NLRs that function as negative regulators of inflammation have been identified based on their abilities to attenuate NF-κB signaling. NLRP12 (Monarch-1) is a prototypical member of this sub-group that negatively regulates both canonical and noncanonical NF-κB signaling in biochemical assays and in colitis and colon cancer models. The role of NLRP12 in infectious diseases has not been extensively studied. Here, we characterized the innate immune response of Nlrp12−/− mice following airway exposure to LPS, Klebsiella pneumoniae and Mycobacterium tuberculosis. In response to E. coli LPS, Nlrp12−/− mice showed a slight decrease in IL-1β and increase in IL-6 production, but these levels were not statistically significant. During K. pneumoniae infection, we observed subtle differences in cytokine levels and significantly reduced numbers of monocytes and lymphocytes in Nlrp12−/− mice. However, the physiological relevance of these findings is unclear as no overt differences in the development of lung disease were observed in the Nlrp12−/− mice. Likewise, Nlrp12−/− mice demonstrated pathologies similar to those observed in the wild type mice following M. tuberculosis infection. Together, these data suggest that NLRP12 does not significantly contribute to the in vivo host innate immune response to LPS stimulation, Klebsiella pneumonia infection or Mycobacterium tuberculosis.

Summary Mycobacterium tuberculosis proteins that are exported out of the bacterial cytoplasm are ideally positioned to be virulence factors; however, the functions of individual exported proteins remain largely unknown. Previous studies identified Rv0199 as an exported membrane protein of unknown function. Here, we characterized the role of Rv0199 in M. tuberculosis virulence using an aerosol model of murine infection. Rv0199 appears to be a member of a Mce-associated membrane (Mam) protein family leading us to rename it OmamA, for orphaned Mce-associated membrane protein A. Consistent with a role in Mce transport, we showed OmamA is required for cholesterol import, which is a Mce4-dependent process. We further demonstrated a function for OmamA in stabilizing protein components of the Mce1 transporter complex. These results indicate a function of OmamA in multiple Mce transporters and one that may be analogous to the role of VirB8 in stabilizing Type IV secretion systems, as structural similarities between Mam proteins and VirB8 proteins are predicted by the Phyre 2 program. In this study, we provide functional information about OmamA and shed light on the function of Mam family proteins in Mce transporters.