Macrophage infectivity potentiator protein, a peptidyl prolyl cis-trans isomerase, essential for Coxiella burnetii growth and pathogenesis.

Coxiella burnetii is a Gram-negative intracellular pathogen that causes the debilitating disease Q fever, which affects both animals and humans. The only available human vaccine, Q-Vax, is effective but has a high risk of severe adverse reactions, limiting its use as a countermeasure to contain outbreaks. Therefore, it is essential to identify new drug targets to treat this infection. Macrophage infectivity potentiator (Mip) proteins catalyse the folding of proline-containing proteins through their peptidyl prolyl cis-trans isomerase (PPIase) activity and have been shown to play an important role in the virulence of several pathogenic bacteria. To date the role of the Mip protein in C. burnetii pathogenesis has not been investigated. This study demonstrates that CbMip is likely to be an essential protein in C. burnetii. The pipecolic acid derived compounds, SF235 and AN296, which have shown utility in targeting other Mip proteins from pathogenic bacteria, demonstrate inhibitory activities against CbMip. These compounds were found to significantly inhibit intracellular replication of C. burnetii in both HeLa and THP-1 cells. Furthermore, SF235 and AN296 were also found to exhibit antibiotic properties against both the virulent (Phase I) and avirulent (Phase II) forms of C. burnetii Nine Mile Strain in axenic culture. Comparative proteomics, in the presence of AN296, revealed alterations in stress responses with H2O2 sensitivity assays validating that Mip inhibition increases the sensitivity of C. burnetii to oxidative stress. In addition, SF235 and AN296 were effective in vivo and significantly improved the survival of Galleria mellonella infected with C. burnetii. These results suggest that unlike in other bacteria, Mip in C. burnetii is required for replication and that the development of more potent inhibitors against CbMip is warranted and offer potential as novel therapeutics against this pathogen.

Proteomic Identification of Coxiella burnetii Effector Proteins Targeted to the Host Cell Mitochondria During Infection.

Modulation of the host cell is integral to the survival and replication of microbial pathogens. Several intracellular bacterial pathogens deliver bacterial proteins, termed "effector proteins" into the host cell during infection by sophisticated protein translocation systems, which manipulate cellular processes and functions. The functional contribution of individual effectors is poorly characterized, particularly in intracellular bacterial pathogens with large effector protein repertoires. Technical caveats have limited the capacity to study these proteins during a native infection, with many effector proteins having only been demonstrated to be translocated during over-expression of tagged versions. Here, we developed a novel strategy to examine effector proteins in the context of infection. We coupled a broad, unbiased proteomics-based screen with organelle purification to study the host-pathogen interactions occurring between the host cell mitochondrion and the Gram-negative, Q fever pathogen Coxiella burnetii. We identify four novel mitochondrially-targeted C. burnetii effector proteins, renamed Mitochondrial Coxiella effector protein (Mce) B to E. Examination of the subcellular localization of ectopically expressed proteins confirmed their mitochondrial localization, demonstrating the robustness of our approach. Subsequent biochemical analysis and affinity enrichment proteomics of one of these effector proteins, MceC, revealed the protein localizes to the inner membrane and can interact with components of the mitochondrial quality control machinery. Our study adapts high-sensitivity proteomics to study intracellular host-pathogen interactions, providing a robust strategy to examine the subcellular localization of effector proteins during native infection. This approach could be applied to a range of pathogens and host cell compartments to provide a rich map of effector dynamics throughout infection.

Biogenesis of the Spacious Coxiella-Containing Vacuole Depends on Host Transcription Factors TFEB and TFE3.

Coxiella burnetii is an obligate intracellular bacterial pathogen that replicates inside the lysosome-derived Coxiella-containing vacuole (CCV). To establish this unique niche, C. burnetii requires the Dot/Icm type IV secretion system (T4SS) to translocate a cohort of effector proteins into the host cell, which modulate multiple cellular processes. To characterize the host-pathogen interactions that occur during C. burnetii infection, stable-isotope labeling by amino acids in cell culture (SILAC)-based proteomics was used to identify changes in the host proteome during infection of a human-derived macrophage cell line. These data revealed that the abundances of many proteins involved in host cell autophagy and lysosome biogenesis were increased in infected cells. Thus, the role of the host transcription factors TFEB and TFE3, which regulate the expression of a network of genes involved in autophagy and lysosomal biogenesis, were examined in the context of C. burnetii infection. During infection with C. burnetii, both TFEB and TFE3 were activated, as demonstrated by the transport of these proteins from the cytoplasm into the nucleus. The nuclear translocation of these transcription factors was shown to be dependent on the T4SS, as a Dot/Icm mutant showed reduced nuclear translocation of TFEB and TFE3. This was supported by the observation that blocking bacterial translation with chloramphenicol resulted in the movement of TFEB and TFE3 back into the cytoplasm. Silencing of the TFEB and TFE3 genes, alone or in combination, significantly reduced the size of the CCV, which indicates that these host transcription factors facilitate the expansion and maintenance of the organelle that supports C. burnetii intracellular replication.

Genetic Manipulation of Non-pneumophila Legionella: Protocols Developed for Legionella longbeachae.

Current biomedical research into Legionnaires' disease is dominated by studies of Legionella pneumophila, largely because this pathogen is responsible for approximately 90% of clinical disease worldwide. However, in certain geographical regions, infections with non-pneumophila species are responsible for a significant proportion of diagnosed Legionnaires' disease. Understanding the pathogenesis of these non-pneumophila species of Legionella is an important step toward clinical intervention. The capacity to genetically manipulate these pathogens is essential in order to understand the genetic factors that contribute to infection and the environmental life cycle of these bacteria. The capacity to delete, mutate, and relocate genetic regions of interest allows molecular research into gene function and importance. In this chapter, methods are outlined to introduce plasmids into Legionella by electroporation. This technique is particularly useful as it is often the essential preliminary step to experiments that observe the behavior of the bacterium under altered conditions, for example, the transformation of bacteria with reporter plasmids to monitor Dot/Icm effector translocation. Electroporation is a well-established method for transformation of competent bacteria, and here specific protocols are provided, suiting a range of materials and conditions that have been successfully applied to L. longbeachae and L. dumoffii. Additionally, a homologous recombination approach to delete genetic regions of interest in L. longbeachae is outlined. The application of these techniques allows for identification of the genetic determinants of non-pneumophila Legionella virulence and for important comparative studies with other Legionella species.

De novo NAD synthesis is required for intracellular replication of Coxiella burnetii, the causative agent of the neglected zoonotic disease Q fever.

Coxiella burnetii is an intracellular Gram-negative bacterium responsible for the important zoonotic disease Q fever. Improved genetic tools and the ability to grow this bacterium in host cell-free media has advanced the study of C. burnetii pathogenesis, but the mechanisms that allow it to survive inside the hostile phagolysosome remain incompletely understood. Previous screening of a transposon mutant library for replication within HeLa cells has suggested that nadB, encoding a putative l-aspartate oxidase required for de novo NAD synthesis, is needed for intracellular replication. Here, using genetic complementation of two independent nadB mutants and intracellular replication assays, we confirmed this finding. Untargeted metabolite analyses demonstrated key changes in metabolites in the NAD biosynthetic pathway in the nadB mutant compared with the WT, confirming the involvement of NadB in de novo NAD synthesis. Bioinformatic analysis revealed the presence of a functionally conserved arginine residue at position 275. Using site-directed mutagenesis to substitute this residue with leucine, which abolishes the activity of Escherichia coli NadB, and expression of WT and R275L GST-NadB fusion proteins in E. coli JM109, we found that purified recombinant WT GST-NadB has l-aspartate oxidase activity and that the R275L NadB variant is inactive. Complementation of the C. burnetii nadB mutant with a plasmid expressing this inactive R275L NadB failed to restore replication to WT levels, confirming the link between de novo NAD synthesis and intracellular replication of C. burnetii This suggests that targeting this prokaryotic-specific pathway could advance the development of therapeutics to combat C. burnetii infections.

A Farnesylated Coxiella burnetii Effector Forms a Multimeric Complex at the Mitochondrial Outer Membrane during Infection.

Coxiella burnetii, the causative agent of Q fever, establishes a unique lysosome-derived intracellular niche termed the Coxiella-containing vacuole (CCV). The Dot/Icm-type IVB secretion system is essential for the biogenesis of the CCV and the intracellular replication of Coxiella Effector proteins, translocated into the host cell through this apparatus, act to modulate host trafficking and signaling processes to facilitate CCV development. Here we investigated the role of CBU0077, a conserved Coxiella effector that had previously been observed to localize to lysosomal membranes. CBU0077 was dispensable for the intracellular replication of Coxiella in HeLa and THP-1 cells and did not appear to participate in CCV biogenesis. Intriguingly, native and epitope-tagged CBU0077 produced by Coxiella displayed specific punctate localization at host cell mitochondria. As such, we designated CBU0077 MceA (mitochondrial Coxiellaeffector protein A). Analysis of ectopically expressed MceA truncations revealed that the capacity to traffic to mitochondria is encoded within the first 84 amino acids of this protein. MceA is farnesylated by the host cell; however, this does not impact mitochondrial localization. Examination of mitochondria isolated from infected cells revealed that MceA is specifically integrated into the mitochondrial outer membrane and forms a complex of approximately 120 kDa. Engineering Coxiella to express either MceA tagged with 3×FLAG or MceA tagged with 2×hemagglutinin allowed us to perform immunoprecipitation experiments that showed that MceA forms a homo-oligomeric species at the mitochondrial outer membrane during infection. This research reveals that mitochondria are a bona fide target of Coxiella effectors and MceA is a complex-forming effector at the mitochondrial outer membrane during Coxiella infection.

The Effector Cig57 Hijacks FCHO-Mediated Vesicular Trafficking to Facilitate Intracellular Replication of Coxiella burnetii.

Coxiella burnetii is an intracellular bacterial pathogen that infects alveolar macrophages and replicates within a unique lysosome-derived vacuole. When Coxiella is trafficked to a host cell lysosome the essential Dot/Icm type IV secretion system is activated allowing over 130 bacterial effector proteins to be translocated into the host cytosol. This cohort of effectors is believed to manipulate host cell functions to facilitate Coxiella-containing vacuole (CCV) biogenesis and bacterial replication. Transposon mutagenesis has demonstrated that the Dot/Icm effector Cig57 is required for CCV development and intracellular replication of Coxiella. Here, we demonstrate a role for Cig57 in subverting clathrin-mediated traffic through its interaction with FCHO2, an accessory protein of clathrin coated pits. A yeast two-hybrid screen identified FCHO2 as a binding partner of Cig57 and this interaction was confirmed during infection using immunoprecipitation experiments. The interaction between Cig57 and FCHO2 is dependent on one of three endocytic sorting motif encoded by Cig57. Importantly, complementation analysis demonstrated that this endocytic sorting motif is required for full function of Cig57. Consistent with the intracellular growth defect in cig57-disrupted Coxiella, siRNA gene silencing of FCHO2 or clathrin (CLTC) inhibits Coxiella growth and CCV biogenesis. Clathrin is recruited to the replicative CCV in a manner that is dependent on the interaction between Cig57 and FCHO2. Creation of an FCHO2 knockout cell line confirmed the importance of this protein for CCV expansion, intracellular replication of Coxiella and clathrin recruitment to the CCV. Collectively, these results reveal Cig57 to be a significant virulence factor that co-opts clathrin-mediated trafficking, via interaction with FCHO2, to facilitate the biogenesis of the fusogenic Coxiella replicative vacuole and enable intracellular success of this human pathogen.

Impact of a Cross-Kingdom Signaling Molecule of Candida albicans on Acinetobacter baumannii Physiology.

Multidrug-resistant (MDR) Acinetobacter baumannii is an opportunistic human pathogen that has become highly problematic in the clinical environment. Novel therapies are desperately required. To assist in identifying new therapeutic targets, the antagonistic interactions between A. baumannii and the most common human fungal pathogen, Candida albicans, were studied. We have observed that the C. albicans quorum-sensing molecule, farnesol, has cross-kingdom interactions, affecting the viability of A. baumannii. To gain an understanding of its mechanism, the transcriptional profile of A. baumannii exposed to farnesol was examined. Farnesol caused dysregulation of a large number of genes involved in cell membrane biogenesis, multidrug efflux pumps (AcrAB-like and AdeIJK-like), and A. baumannii virulence traits such as biofilm formation (csuA, csuB, and ompA) and motility (pilZ and pilH). We also observed a strong induction in genes involved in cell division (minD, minE, ftsK, ftsB, and ftsL). These transcriptional data were supported by functional assays showing that farnesol disrupts A. baumannii cell membrane integrity, alters cell morphology, and impairs virulence characteristics such as biofilm formation and twitching motility. Moreover, we showed that A. baumannii uses efflux pumps as a defense mechanism against this eukaryotic signaling molecule. Owing to its effects on membrane integrity, farnesol was tested to see if it potentiated the activity of the membrane-acting polymyxin antibiotic colistin. When coadministered, farnesol increased sensitivity to colistin for otherwise resistant strains. These data provide mechanistic understanding of the antagonistic interactions between diverse pathogens and may provide important insights into novel therapeutic strategies.

Inhibition of inflammasome activation by Coxiella burnetii type IV secretion system effector IcaA.

Coxiella burnetii is a highly infectious bacterium that promotes its own replication in macrophages by inhibiting several host cell responses. Here, we show that C. burnetii inhibits caspase-1 activation in primary mouse macrophages. By using co-infection experiments, we determine that the infection of macrophages with C. burnetii inhibits the caspase-11-mediated non-canonical activation of the NLRP3 inflammasome induced by subsequent infection with Escherichia coli or Legionella pneumophila. Genetic screening using flagellin mutants of L. pneumophila as a surrogate host, reveals a novel C. burnetii gene (IcaA) involved in the inhibition of caspase activation. Expression of IcaA in L. pneumophila inhibited the caspase-11 activation in macrophages. Moreover, icaA(-) mutants of C. burnetii failed to suppress the caspase-11-mediated inflammasome activation induced by L. pneumophila. Our data reveal IcaA as a novel C. burnetii effector protein that is secreted by the Dot/Icm type IV secretion system and interferes with the caspase-11-induced, non-canonical activation of the inflammasome.

The use of high-throughput DNA sequencing in the investigation of antigenic variation: application to Neisseria species.

Antigenic variation occurs in a broad range of species. This process resembles gene conversion in that variant DNA is unidirectionally transferred from partial gene copies (or silent loci) into an expression locus. Previous studies of antigenic variation have involved the amplification and sequencing of individual genes from hundreds of colonies. Using the pilE gene from Neisseria gonorrhoeae we have demonstrated that it is possible to use PCR amplification, followed by high-throughput DNA sequencing and a novel assembly process, to detect individual antigenic variation events. The ability to detect these events was much greater than has previously been possible. In N. gonorrhoeae most silent loci contain multiple partial gene copies. Here we show that there is a bias towards using the copy at the 3' end of the silent loci (copy 1) as the donor sequence. The pilE gene of N. gonorrhoeae and some strains of Neisseria meningitidis encode class I pilin, but strains of N. meningitidis from clonal complexes 8 and 11 encode a class II pilin. We have confirmed that the class II pili of meningococcal strain FAM18 (clonal complex 11) are non-variable, and this is also true for the class II pili of strain NMB from clonal complex 8. In addition when a gene encoding class I pilin was moved into the meningococcal strain NMB background there was no evidence of antigenic variation. Finally we investigated several members of the opa gene family of N. gonorrhoeae, where it has been suggested that limited variation occurs. Variation was detected in the opaK gene that is located close to pilE, but not at the opaJ gene located elsewhere on the genome. The approach described here promises to dramatically improve studies of the extent and nature of antigenic variation systems in a variety of species.

Transcriptional response of Leptospira interrogans to iron limitation and characterization of a PerR homolog.

Leptospirosis is a globally significant zoonosis caused by Leptospira spp. Iron is essential for growth of most bacterial species. Since iron availability is low in the host, pathogens have evolved complex iron acquisition mechanisms to survive and establish infection. In many bacteria, expression of iron uptake and storage proteins is regulated by Fur. L. interrogans encodes four predicted Fur homologs; we have constructed a mutation in one of these, la1857. We conducted microarray analysis to identify iron-responsive genes and to study the effects of la1857 mutation on gene expression. Under iron-limiting conditions, 43 genes were upregulated and 49 genes were downregulated in the wild type. Genes encoding proteins with predicted involvement in inorganic ion transport and metabolism (including TonB-dependent proteins and outer membrane transport proteins) were overrepresented in the upregulated list, while 54% of differentially expressed genes had no known function. There were 16 upregulated genes of unknown function which are absent from the saprophyte L. biflexa and which therefore may encode virulence-associated factors. Expression of iron-responsive genes was not significantly affected by mutagenesis of la1857, indicating that LA1857 is not a global regulator of iron homeostasis. Upregulation of heme biosynthetic genes and a putative catalase in the mutant suggested that LA1857 is more similar to PerR, a regulator of the oxidative stress response. Indeed, the la1857 mutant was more resistant to peroxide stress than the wild type. Our results provide insights into the role of iron in leptospiral metabolism and regulation of the oxidative stress response, including genes likely to be important for virulence.