carP, encoding a Ca2+-regulated putative phytase, is evolutionarily conserved in Pseudomonas aeruginosa and has potential as a biomarker.

Pseudomonas aeruginosa infects patients with cystic fibrosis, burns, wounds and implants. Previously, our group showed that elevated Ca2+ positively regulates the production of several virulence factors in P. aeruginosa, such as biofilm formation, production of pyocyanin and secreted proteases. We have identified a Ca2+-regulated ß-propeller putative phytase, CarP, which is required for Ca2+ tolerance, regulation of the intracellular Ca2+ levels, and plays a role in Ca2+ regulation of P. aeruginosa virulence. Here, we studied the conservation of carP sequence and its occurrence in diverse phylogenetic groups of bacteria. In silico analysis revealed that carP and its two paralogues PA2017 and PA0319 are primarily present in P. aeruginosa and belong to the core genome of the species. We identified 155 single nucleotide alterations within carP, 42 of which lead to missense mutations with only three that affected the predicted 3D structure of the protein. PCR analyses with carP-specific primers detected P. aeruginosa specifically in 70 clinical and environmental samples. Sequence comparison demonstrated that carP is overall highly conserved in P. aeruginosa isolated from diverse environments. Such evolutionary preservation of carP illustrates its importance for P. aeruginosa adaptations to diverse environments and demonstrates its potential as a biomarker.

Chlamydia trachomatis inclusion membrane protein MrcA interacts with the inositol 1,4,5-trisphosphate receptor type 3 (ITPR3) to regulate extrusion formation.

Chlamydia trachomatis is an obligate intracellular bacterium that replicates within a vacuole termed an inclusion. At the end of their intracellular developmental cycle, chlamydiae are released either by lysis of the host cell or extrusion of the intact inclusion. The inclusion membrane is extensively modified by the insertion of type III secreted inclusion membrane proteins, Incs, which contribute to inclusion membrane structure and facilitate host-pathogen interactions. An interaction was identified between the inclusion membrane protein, MrcA, and the Ca2+ channel inositol-1,4,5-trisphosphate receptor, type 3 (ITPR3). ITPR3 was recruited and localized to active Src-family-kinase rich microdomains on the inclusion membrane as was the Ca2+ sensor, STIM1. Disruption of MrcA by directed mutagenesis resulted in loss of ITPR3 recruitment and simultaneous reduction of chlamydial release by extrusion. Complementation of MrcA restored ITPR3 recruitment and extrusion. Inhibition of extrusion was also observed following siRNA depletion of host ITPR3 or STIM1. Chlamydial extrusion was also inhibited by the calcium chelator BAPTA-AM. Each of these treatments resulted in a concomitant reduction in phosphorylation of the myosin regulatory light chain (MLC2) and a loss of myosin motor activity at the end of the developmental cycle which is consistent with the reduced extrusion formation. These studies suggest that Ca2+ signaling pathways play an important role in regulation of release mechanisms by C. trachomatis.

Comparative genome sequencing of Rickettsia rickettsii strains that differ in virulence.

Rickettsia rickettsii is an obligate intracellular pathogen that is the causative agent of Rocky Mountain spotted fever. Strains of R. rickettsii differ dramatically in virulence. In a guinea pig model of infection, the severity of disease as assessed by fever response varies from the most virulent, Sheila Smith, to Iowa, which causes no fever. To identify potential determinants of virulence in R. rickettsii, the genomes of two additional strains were sequenced for comparison to known sequences (comparative genome sequencing [CGS]). R. rickettsii Morgan and R strains were compared to the avirulent R. rickettsii Iowa and virulent R. rickettsii Sheila Smith strains. The Montana strains Sheila Smith and R were found to be highly similar while the eastern strains Iowa and Morgan were most similar to each other. A major surface antigen, rickettsial outer membrane protein A (rOmpA), is severely truncated in the Iowa strain. The region of ompA containing 13 tandem repeats was sequenced, revealing only seven shared SNPs (four nonsynonymous) for R and Morgan strains compared to Sheila Smith, with an additional 17 SNPs identified in Morgan. Another major surface antigen and autotransporter, rOmpB, exhibits a defect in processing in the Iowa strain such that the beta fragment is not cleaved. Sequence analysis of ompB reveals identical sequences between Iowa and Morgan strains and between the R and Sheila Smith strains. The number of SNPs and insertions/deletions between sequences of the two Montana strains and the two eastern strains is low, thus narrowing the field of possible virulence factors.

Chlamydia trachomatis inclusion membrane protein CT850 interacts with the dynein light chain DYNLT1 (Tctex1).

Chlamydia trachomatis actively subverts the minus-end directed microtubule motor, dynein, to traffic along microtubule tracks to the Microtubule Organizing Center (MTOC) where it remains within a membrane bound replicative vacuole for the duration of its intracellular development. Unlike most substrates of the dynein motor, disruption of the dynactin cargo-linking complex by over-expression of the p50 dynamitin subunit does not inhibit C. trachomatis transport. A requirement for chlamydial protein synthesis to initiate this process suggests that a chlamydial product supersedes a requirement for p50 dynamitin. A yeast 2-hybrid system was used to screen the chlamydia inclusion membrane protein CT850 against a HeLa cell cDNA library and identified an interaction with the dynein light chain DYNLT1 (Tctex1). This interaction was at least partially dependent upon an (R/K-R/K-X-X-R/K) motif that is characteristic of DYNLT1 binding domains. CT850 expressed ectopically in HeLa cells localized at the MTOC and this localization is similarly dependent upon the predicted DYNLT1 binding domain. Furthermore, DYNLT1 is enriched at focal concentrations of CT850 on the chlamydial inclusion membrane that are known to interact with dynein and microtubules. Depletion of DYNLT1 disrupts the characteristic association of the inclusion membrane with centrosomes. Collectively, the results suggest that CT850 interacts with DYNLT1 to promote appropriate positioning of the inclusion at the MTOC.

EF-Hand Calcium Sensor, EfhP, Controls Transcriptional Regulation of Iron Uptake by Calcium in Pseudomonas aeruginosa.

The human pathogen Pseudomonas aeruginosa poses a major risk for a range of severe infections, particularly lung infections in patients suffering from cystic fibrosis (CF). As previously reported, the virulent behavior of this pathogen is enhanced by elevated levels of Ca 2+ that are commonly present in CF nasal and lung fluids. In addition, a Ca 2+ -binding EF-hand protein, EfhP (PA4107), was partially characterized and shown to be critical for the Ca 2+ -regulated virulence in P. aeruginosa . Here we describe the rapid (10 min, 60 min), and adaptive (12 h) transcriptional responses of PAO1 to elevated Ca 2+ detected by genome-wide RNA sequencing and show that efhP deletion significantly hindered both rapid and adaptive Ca 2+ regulation. The most differentially regulated genes included multiple Fe sequestering mechanisms, a large number of extracytoplasmic function sigma factors (ECFσ) and several virulence factors, such as production of pyocins. The Ca 2+ regulation of Fe uptake was also observed in CF clinical isolates and appeared to involve the global regulator Fur. In addition, we showed that the efhP transcription is controlled by Ca 2+ and Fe, and this regulation required Ca 2+ -dependent two-component regulatory system CarSR. Furthermore, the efhP expression is significantly increased in CF clinical isolates and upon pathogen internalization into epithelial cells. Overall, the results established for the first time that Ca 2+ controls Fe sequestering mechanisms in P. aeruginosa and that EfhP plays a key role in the regulatory interconnectedness between Ca 2+ and Fe signaling pathways, the two distinct and important signaling pathways that guide the pathogen's adaptation to host. IMPORTANCE: Pseudomonas aeruginosa ( Pa ) poses a major risk for severe infections, particularly in patients suffering from cystic fibrosis (CF). For the first time, kinetic RNA sequencing analysis identified Pa rapid and adaptive transcriptional responses to Ca 2+ levels consistent with those present in CF respiratory fluids. The most highly upregulated processes include iron sequestering, iron starvation sigma factors, and self-lysis factors pyocins. An EF-hand Ca 2+ sensor, EfhP, is required for at least 1/3 of the Ca 2+ response, including all the iron uptake mechanisms and production of pyocins. Transcription of efhP itself is regulated by Ca 2+ , Fe, and increases during interactions with host epithelial cells, suggesting the protein's important role in Pa infections. The findings establish the regulatory interconnectedness between Ca 2+ and iron signaling pathways that shape Pa transcriptional responses. Therefore, understanding Pa's transcriptional response to Ca 2+ and associated regulatory mechanisms will serve the development of future therapeutics targeting Pa dangerous infections.

Lethality and cooperation of Pseudomonas aeruginosa quorum-sensing mutants in Drosophila melanogaster infection models.

The virulence profiles of Pseudomonas aeruginosa quorum-sensing (QS) mutants were assessed in Drosophila melanogaster feeding and nicking infection models. Functional RhlIR and LasIR QS systems were required for killing in the fly feeding infection model but were not essential in the fly nicking infection model. Mixed infections between PAO1 and strains harbouring mutations in lasR, rhlI and lasI rhlI resulted in increased lethality in the fly feeding model compared with either isolate alone. These results suggested that the parental strain could cooperate with QS mutants in the Drosophila feeding infection model. Finally, the mixed infection between PAO1 and an rhlR mutant resulted in spiteful behaviour and reduced pathogenicity of the mixed culture.

Evolution and conservation of predicted inclusion membrane proteins in chlamydiae.

Chlamydia spp. are obligate intracellular pathogens that replicate within a vacuole termed the inclusion. Chlamydiae extensively modify the inclusion membrane via the insertion of chlamydial inclusion membrane proteins (Incs) which decorate the cytosolic face of the inclusion. We have assessed the overall relatedness and phylogeny of Incs in order to identify potential evolutionary trends. Despite a high degree of conservation among Incs within C. trachomatis serovars, phylogenetic analysis showed that some Incs cluster according to clinical groupings suggesting that certain Incs may contribute to tissue tropism. Bioinformatic predictions identified Incs in five chlamydial species: 55 in C. trachomatis, 68 in C. felis, 92 in C. pneumoniae, 79 in C. caviae, and 54 in C. muridarum. Inc homologues were compared between chlamydial species and 23 core Incs were identified as shared among all species. Genomic expansion of Incs was identified in C. pneumoniae, C. caviae, and C. felis but not C. trachomatis or C. muridarum.

Eumelanin-Inspired Antimicrobial with Biocidal Activity against Methicillin-Resistant Staphylococcus aureus.

The reliance on antibiotics and antimicrobials to treat bacterial infectious diseases is threatened by the emergence of antibiotic resistance and multi-drug-resistant organisms, thus having the potential to greatly impact human health. Thus, the discovery and development of antimicrobials capable of acting on antibiotic-resistant bacteria is a major area of significance in scientific research. Herein, we present the development of a eumelanin-inspired antimicrobial capable of killing methicillin-resistant Staphylococcus aureus (MRSA). By ligating quaternary ammonium-functionalized "arms" to a eumelanin-inspired indole with intrinsic antimicrobial activity, an antimicrobial agent with enhanced activity was prepared. This resulting antimicrobial, EIPE-1, had a minimum inhibitory concentration of 16 µg/mL (17.1 µM) against a clinical isolate of MRSA obtained from an adult cystic fibrosis patient. The biocidal activity occurred within 30 min of exposure and resulted in changes to the bacterial cell surface as visualized with a scanning electron microscope. Taken together, these studies demonstrate that EIPE-1 is effective at killing MRSA.

Corrigendum: Genetic inactivation of Chlamydia trachomatis inclusion membrane protein CT228 alters MYPT1 recruitment, extrusion production, and longevity of infection.

[This corrects the article DOI: 10.3389/fcimb.2018.00415.].

Transformation frequency of a mariner-based transposon in Rickettsia rickettsii.

Transformation frequencies of a mariner-based transposon system in Rickettsia rickettsii were determined using a plaque assay system for enumeration and isolation of mutants. Sequence analysis of insertion sites in both R. rickettsii and R. prowazekii indicated that insertions were random. Transposon mutagenesis provides a useful tool for rickettsial research.

Disruption of the Rickettsia rickettsii Sca2 autotransporter inhibits actin-based motility.

Rickettsii rickettsii, the etiologic agent of Rocky Mountain spotted fever, replicates within the cytosol of infected cells and uses actin-based motility to spread inter- and intracellularly. Although the ultrastructure of the actin tail and host proteins associated with it are distinct from those of Listeria or Shigella, comparatively little is known regarding the rickettsial proteins involved in its organization. Here, we have used random transposon mutagenesis of R. rickettsii to generate a small-plaque mutant that is defective in actin-based motility and does not spread directly from cell to cell as is characteristic of spotted fever group rickettsiae. The transposon insertion site of this mutant strain was within Sca2, a member of a family of large autotransporter proteins. Sca2 exhibits several features suggestive of its apparent role in actin-based motility. It displays an N-terminal secretory signal peptide, a C-terminal predicted autotransporter domain, up to four predicted Wasp homology 2 (WH2) domains, and two proline-rich domains, one with similarity to eukaryotic formins. In a guinea pig model of infection, the Sca2 mutant did not elicit fever, suggesting that Sca2 and actin-based motility are virulence factors of spotted fever group rickettsiae.

Phylogenetic analysis of Chlamydia trachomatis Tarp and correlation with clinical phenotype.

Chlamydia trachomatis is the leading cause of infectious blindness worldwide and is the most commonly reported pathogen causing sexually transmitted infections. Tarp (translocated actin recruiting phosphoprotein), a type III secreted effector that mediates actin nucleation, is central to C. trachomatis infection. The phylogenetic analysis of tarP from reference strains as well as ocular, genital, and lymphogranuloma venereum (LGV) clinical isolates demonstrated an evolutionary relationship with disease phenotype, with LGV and ocular isolates branched into clades that were separate from the urogenital isolates. The sequence analysis of Tarp indicated a high degree of variability and identified trends within clinical groupings. Tarps from LGV strains contained the highest number of tyrosine-rich repeat regions (up to nine) and the fewest (two) predicted actin binding domains. The converse was noted for Tarp proteins from ocular isolates that contained up to four actin binding domains and as few as one tyrosine-rich repeat region. The results suggest that Tarp is among the few known genes to play a role in C. trachomatis adaptations to specific niches within the host.

Hijacking and Use of Host Kinases by Chlamydiae.

Chlamydia species are causative agents of sexually transmitted infections, blinding trachoma, and animal infections with zoonotic potential. Being an obligate intracellular pathogen, Chlamydia relies on the host cell for its survival and development, subverting various host cell processes throughout the infection cycle. A key subset of host proteins utilized by Chlamydia include an assortment of host kinase signaling networks which are vital for many chlamydial processes including entry, nutrient acquisition, and suppression of host cell apoptosis. In this review, we summarize the recent advancements in our understanding of host kinase subversion by Chlamydia.

Chlamydia trachomatis recruits protein kinase C during infection.

Chlamydia trachomatis is a significant pathogen with global and economic impact. As an obligate intracellular pathogen, C. trachomatis resides inside the inclusion, a parasitophorous vacuole, and depends on the host cell for survival and transition through a biphasic development cycle. During infection, C. trachomatis is known to manipulate multiple signaling pathways and recruit an assortment of host proteins to the inclusion membrane, including host kinases. Here, we show recruitment of multiple isoforms of protein kinase C (PKC) including active phosphorylated PKC isoforms to the chlamydial inclusion colocalizing with active Src family kinases. Pharmacological inhibition of PKC led to a modest reduction of infectious progeny production. PKC phosphorylated substrates were seen recruited to the entire periphery of the inclusion membrane. Infected whole cell lysates showed altered PKC phosphorylation of substrates during the course of infection. Assessment of different chlamydial species showed recruitment of PKC and PKC phosphorylated substrates were limited to C. trachomatis. Taken together, PKC and PKC substrate recruitment may provide significant insights into how C. trachomatis manipulates multiple host signaling cascades during infection.

Pseudomonas aeruginosa cystic fibrosis isolates from individual patients demonstrate a range of levels of lethality in two Drosophila melanogaster infection models.

Recently, two Drosophila melanogaster models of infection, fly feeding and fly nicking, have been developed that allow a determination of pathogenic potential of Pseudomonas aeruginosa isolates. In this study, control strains, isolates from burn wounds, and isolates from the sputa of cystic fibrosis (CF) patients were used to compare the two infection models to determine whether any of the isolates might be better adapted to either of the models. In addition, our goal was to determine the variability of isolates from individual CF patients. Three of four control strains (PAO1, PAK, and PA14) caused significant mortality in the flies in both models of infection. The remaining control strain, PA103, was lethal to flies in the nicking model but lacked significant lethality in the feeding model. The burn wound isolates had a high level of lethality in both models. Interestingly, the CF isolates had the largest diversity of lethality in both models of infection. The range of pathogenic potentials of the CF isolates occurred across a cohort of patients, both at the patient level and down to the level of individual sputum samples. The majority of all isolates had similar levels of lethality in both fly infection models. However, two CF isolates were significantly more lethal in the nicking model, and three CF isolates were significantly more lethal in the feeding model. In conclusion, the two Drosophila infection models were useful for the analysis of the diversity of pathogenic potentials of P. aeruginosa isolates.

Genetic Inactivation of Chlamydia trachomatis Inclusion Membrane Protein CT228 Alters MYPT1 Recruitment, Extrusion Production, and Longevity of Infection.

Chlamydia trachomatis is an obligate intracellular pathogen with global health and economic impact. Upon infection, C. trachomatis resides within a protective niche, the inclusion, wherein it replicates and usurps host cell machinery and resources. The inclusion membrane is the key host-pathogen interface that governs specific protein-protein interactions to manipulate host signaling pathways. At the conclusion of the infection cycle, C. trachomatis exits the host cell via lysis or extrusion. Extrusion depends on the phosphorylation state of myosin light chain 2 (MLC2); the extent of phosphorylation is determined by the ongoing opposing activities of myosin phosphatase (MYPT1) and myosin kinase (MLCK). Previously, it was shown that MYPT1 is recruited to the inclusion and interacts with CT228 for regulation of host cell egress. In this study, we generated a targeted chromosomal mutation of CT228 (L2-ΔCT228) using the TargeTron system and demonstrate a loss of MYPT1 recruitment and increase in extrusion production in vitro. Mutation of CT228 did not affect chlamydial growth in cell culture or recruitment of MLC2. Moreover, we document a delay in clearance of L2-ΔCT228 during murine intravaginal infection as well as a reduction in systemic humoral response, relative to L2-wild type. Taken together, the data suggest that loss of MYPT1 recruitment (as a result of CT228 disruption) regulates the degree of host cell exit via extrusion and affects the longevity of infection in vivo.

The Coxiella Burnetii type IVB secretion system (T4BSS) component DotA is released/secreted during infection of host cells and during in vitro growth in a T4BSS-dependent manner.

Coxiella burnetii is a Gram-negative intracellular pathogen and is the causative agent of the zoonotic disease Q fever. To cause disease, C. burnetii requires a functional type IVB secretion system (T4BSS) to transfer effector proteins required for the establishment and maintenance of a membrane-bound parasitophorous vacuole (PV) and further modulation of host cell process. However, it is not clear how the T4BSS interacts with the PV membrane since neither a secretion pilus nor an extracellular pore forming apparatus has not been described. To address this, we used the acidified citrate cysteine medium (ACCM) along with cell culture infection and immunological techniques to identify the cellular and extracellular localization of T4BSS components. Interestingly, we found that DotA and IcmX were secreted/released in a T4BSS-dependent manner into the ACCM. Analysis of C. burnetii-infected cell lines revealed that DotA colocalized with the host cell marker CD63 (LAMP3) at the PV membrane. In the absence of bacterial protein synthesis, DotA also became depleted from the PV membrane. These data are the first to identify the release/secretion of C. burnetii T4BSS components during axenic growth and the interaction of a T4BSS component with the PV membrane during infection of host cells.

Comparison of Murine Cervicovaginal Infection by Chlamydial Strains: Identification of Extrusions Shed In vivo.

Chlamydia trachomatis is the leading cause of bacterial sexually transmitted infections (STIs) and preventable blindness. Untreated, asymptomatic infection as well as frequent re-infection are common and may drive pelvic inflammatory disease, ectopic pregnancy, and infertility. In vivo models of chlamydial infection continue to be instrumental in progress toward a vaccine and further elucidating the pathogenesis of this intracellular bacterium, however significant gaps in our understanding remain. Chlamydial host cell exit occurs via two mechanisms, lysis and extrusion, although the latter has yet to be reported in vivo and its biological role is unclear. The objective of this study was to investigate whether chlamydial extrusions are shed in vivo following infection with multiple strains of Chlamydia. We utilized an established C3H/HeJ murine cervicovaginal infection model with C. trachomatis serovars D and L2 and the Chlamydia muridarum strain MoPn to monitor the (i) time course of infection and mode of host cell exit, (ii) mucosal and systemic immune response to infection, and (iii) gross and histopathology following clearance of active infection. The key finding herein is the first identification of chlamydial extrusions shed from host cells in an in vivo model. Extrusions, a recently appreciated mode of host cell exit and potential means of dissemination, had been previously observed solely in vitro. The results of this study demonstrate that chlamydial extrusions exist in vivo and thus warrant further investigation to determine their role in chlamydial pathogenesis.

Identification of potential CepR regulated genes using a cep box motif-based search of the Burkholderia cenocepacia genome.

BACKGROUND: The Burkholderia cenocepacia CepIR quorum sensing system has been shown to positively and negatively regulate genes involved in siderophore production, protease expression, motility, biofilm formation and virulence. In this study, two approaches were used to identify genes regulated by the CepIR quorum sensing system. Transposon mutagenesis was used to create lacZ promoter fusions in a cepI mutant that were screened for differential expression in the presence of N-acylhomoserine lactones. A bioinformatics approach was used to screen the B. cenocepacia J2315 genome for CepR binding site motifs. RESULTS: Four positively regulated and two negatively regulated genes were identified by transposon mutagenesis including genes potentially involved in iron transport and virulence. The promoter regions of selected CepR regulated genes and site directed mutagenesis of the cepI promoter were used to predict a consensus cep box sequence for CepR binding. The first-generation consensus sequence for the cep box was used to identify putative cep boxes in the genome sequence. Eight potential CepR regulated genes were chosen and the expression of their promoters analyzed. Six of the eight were shown to be regulated by CepR. A second generation motif was created from the promoters of these six genes in combination with the promoters of cepI, zmpA, and two of the CepR regulated genes identified by transposon mutagenesis. A search of the B. cenocepacia J2315 genome with the new motif identified 55 cep boxes in 65 promoter regions that may be regulated by CepR. CONCLUSION: Using transposon mutagenesis and bioinformatics expression of twelve new genes have been determined to be regulated by the CepIR quorum sensing system. A cep box consensus sequence has been developed based on the predicted cep boxes of ten CepR regulated genes. This consensus cep box has led to the identification of over 50 new genes potentially regulated by the CepIR quorum sensing system.

Draft Genome Sequences of Five Pseudomonas aeruginosa Clinical Strains Isolated from Sputum Samples from Cystic Fibrosis Patients.

We report here the draft genome sequences of five Pseudomonas aeruginosa isolates obtained from sputum samples from two cystic fibrosis patients with chronic colonization. These closely related strains harbor 225 to 493 genes absent from the P. aeruginosa POA1 genome and contain 178 to 179 virulence factors and 29 to 31 antibiotic resistance genes.