Pestilence, persistence and pathogenicity: infection strategies of Bartonella

It has been nearly two decades since the discovery of Bartonella as an agent of bacillary angiomatosis in AIDS patients and persistent bacteremia and \u27nonculturable\u27 endocarditis in homeless people. Since that time, the number of Bartonella species identified has increased from one to 24, and 10 of these bacteria are associated with human disease. Although Bartonella is the only genus that infects human erythrocytes and triggers pathological angiogenesis in the vascular bed, the group remains understudied compared with most other bacterial pathogens. Numerous questions regarding Bartonella\u27s molecular pathogenesis and epidemiology remain unanswered. Virtually every mammal harbors one or more Bartonella species and their transmission typically involves a hematophagous arthropod vector. However, many details regarding epidemiology and the public health threat imposed by these animal reservoirs is unclear. A handful of studies have shown that bartonellae are highly-adapted pathogens whose parasitic strategy has evolved to cause persistent infections of the host. To this end, virulence attributes of Bartonella include the subversion of host cells with effector molecules delivered via a type IV secretion system, induction of pathological angiogenesis through various means, including inhibition of apoptosis and activation of hypoxia-inducing factor 1, use of afimbrial adhesins that are orthologs of Yersinia adhesin A, incorporation of lipopolysaccharides with low endotoxic potency in the outer membrane, and several other virulence factors that help Bartonella infect and persist in erythrocytes and endothelial cells of the host circulatory system

Development of a System for Genetic Manipulation of Bartonella Bacilliformis

Lack of a system for site specific genetic manipulation has severely hindered studies on the molecular biology of all Bartonella species. We report the first site-specific mutagenesis and complementation for a Bartonella species. A highly transformable strain of B. bacilliformis, termed JB584, was isolated and found to exhibit a significant increase in transformation efficiency with the broad-host-range plasmid pBBR1MCS-2, relative to wild-type strains. Restriction analyses of genomic preparations with the methylation-sensitive restriction enzymes ClaI and StuI suggest that strain JB584 possesses a dcm methylase mutation that contributes to its enhanced transformability. A suicide plasmid, pUB1, which contains a polylinker, a pMB1 replicon, and a nptI kanamycin resistance cassette, aas constructed. An internal 508-bp fragment of the B. bacilliformis flagellin gene (fla) was cloned into pUB1 to generate pUB508, a fla-targeting suicide vector. Introduction of pUB508 into JB584 by electroporation generated eight Kan(r) clones of B. bacilliformis, Characterization of one of these strains, termed JB585, indicated that allelic exchange between pUB508 and fla had occurred. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and electron microscopy showed that synthesis of flagellin encoded by fla and secretion/assembly of flagella were abolished. Complementation of fla in trans was accomplished with a pBBR1MCS recombinant containing the entire wild-type fla gene (pBBRFLAG), These data conclusively show that inactivation of fla results in a bald, nonmotile phenotype and that pMB1 and REP replicons make suitable B. bacilliformis suicide and shuttle vectors, respectively. When used in conjunction with the highly transformable strain JB584, this system for site-specific genetic manipulation and complementation provides a new venue for studying the molecular biology of B. bacilliformis

Colonization of \u3ci\u3eLutzomyia verrucarum\u3c/i\u3e and \u3ci\u3eLutzomyia longipalpis\u3c/i\u3e Sand Flies (Diptera: Psychodidae) by \u3ci\u3eBartonella bacilliformis\u3c/i\u3e, the Etiologic Agent of Carrion\u27s Disease

Bartonella bacilliformis is a pathogenic bacterium transmitted to humans presumably by bites of phlebotomine sand flies, infection with which results in a bi-phasic syndrome termed Carrion\u27s disease. After constructing a low-passage GFP-labeled strain of B. bacilliformis, we artificially infected Lutzomyia verrucarum and L. longipalpis populations, and subsequently monitored colonization of sand flies by fluorescence microscopy. Initially, colonization of the two fly species was indistinguishable, with bacteria exhibiting a high degree of motility, yet still confined to the abdominal midgut. After 48h, B. bacilliformis transitioned from bacillus-shape to a non-motile, small coccoid form and appeared to be digested along with the blood meal in both fly species. Differences in colonization patterns became evident at 72h with B. bacilliformis was observed at relatively high density outside the peritrophic membrane in the lumen of the midgut in L. verrucarum, but colonization of L. longipalpis was limited to the blood meal within the intra-peritrophic space of the abdominal midgut, and the majority of bacteria were digested along with the blood meal by day 7. The viability of B. bacilliformis in L. longipalpis was assessed by artificially infecting, homogenizing, and plating for determination of colony-forming units in individual flies over a 13-d time course. Bacteria remained viable at relatively high density for approximately seven days, suggesting that L. longipalpis couple potentially serve as a vector. The capacity of L. longipalpis to transmit viable B. bacilliformis from infected to uninfected meals was analyzed via interrupted feeds. No viable bacteria were retrieved from uninfected blood meals in these experiments. This study provides significant information toward understanding colonization of sand flies by B. bacilliformis and also demonstrates the utility of L. longipalpis as a user-friendly, live-vector model system for studying this severely neglected tropical disease

Pentamidine inhibits Coxiella burnetii growth and 23S rRNA intron splicing in vitro

Coxiella burnetii is the bacterial agent of Q fever in humans. Acute Q fever generally manifests as a flu-like illness and is typically self-resolving. In contrast, chronic Q fever usually presents with endocarditis and is often life-threatening without appropriate antimicrobial therapy. Unfortunately, available options for the successful treatment of chronic Q fever are both limited and protracted (\u3e18 months). Pentamidine, an RNA splice inhibitor used to treat fungal and protozoal infections, was shown to reduce intracellular growth of Coxiella by ca. 73% at a concentration of 1 microM (ca. 0.6 microg/mL) compared with untreated controls, with no detectable toxic effects on host cells. Bacterial targets of pentamidine include Cbu.L1917 and Cbu.L1951, two group I introns that disrupt the 23S rRNA gene of Coxiella, as demonstrated by the drug\u27s ability to inhibit intron RNA splicing in vitro. Since both introns are highly conserved amongst all eight genotypes of the pathogen, pentamidine is predicted to be efficacious against numerous strains of C. burnetii. To our knowledge, this is the first report describing antibacterial activity for this antifungal/antiprotozoal agent

A DNA-Binding Peroxiredoxin of Coxiella Burnetii is Involved in Countering Oxidative Stress During Exponential-Phase Growth

Coxiella burnetii is a Gram-negative, obligate intracellular bacterial pathogen that resides within the harsh, acidic confines of a lysosome-like compartment of the host cell that is termed a parasitophorous vacuole. In this study, we characterized a thiol-specific peroxidase of C. burnetii that belongs to the atypical 2-cysteine subfamily of peroxiredoxins, commonly referred to as bacterioferritin comigratory proteins (BCPs). Coxiella BCP was initially identified as a potential DNA-binding protein by two-dimensional Southwestern (SW) blots of the pathogen\u27s proteome, probed with biotinylated C. burnetii genomic DNA. Confirmation of the identity of the DNA-binding protein as BCP (CBU_0963) was established by matrix-assisted laser desorption ionization tandem time of flight mass spectrometry (MALDI-TOF/TOF MS). Recombinant Coxiella BCP (rBCP) was generated, and its DNA binding was demonstrated by two independent methods, including SW blotting and electrophoretic mobility shift assays (EMSAs). rBCP also demonstrated peroxidase activity in vitro that required thioredoxin-thioredoxin reductase (Trx-TrxR). Both the DNA-binding and peroxidase activities of rBCP were lost upon heat denaturation (100 degrees C, 10 min). Functional expression of Coxiella bcp was demonstrated by trans-complementation of an Escherichia coli bcp mutant, as evidenced by the strain\u27s ability to grow in an oxidative-stress growth medium containing tert-butyl hydroperoxide to levels that were indistinguishable from, or significantly greater than, those observed with its wild-type parental strain and significantly greater than bcp mutant levels (P \u3c 0.05). rBCP was also found to protect supercoiled plasmid DNA from oxidative damage (i.e., nicking) in vitro. Maximal expression of the bcp gene coincided with the pathogen\u27s early (day 2 to 3) exponential-growth phase in an experiment involving synchronized infection of an epithelial (Vero) host cell line. Taken as a whole, the results show that Coxiella BCP binds DNA and likely serves to detoxify endogenous hydroperoxide byproducts of Coxiella\u27s metabolism during intracellular replication

CBU_1932: A Hypothetical DNA-Binding Protein of the Q Fever Pathogen Coxiella Burnetii

Coxiella burnetii is an obligate intracellular bacterial pathogen that resides within a lysosome-like acidic compartment of the eukaryotic host cell and may cause acute and chronic human infections.  Our recent transcriptome analysis of C. burnetii demonstrated that the CBU_1932 open reading frame displayed an exceptionally high transcript level at 11,481 transcripts per million (TPM), well above average transcript quantity for remaining ORFs in the genome.  Due to it’s high transcript level we hypothesize the corresponding protein may play an important role for Coxiella.  Analysis of the CBU_1932 locus indicates that one of the adjacent ORFs, CBU_1933 is a hypothetical DNA binding protein.  The protein encoded by CBU_1932 ORF consists of 66 amino acid residues with an unusually high percentage (42%) of residues being basic, including 20 lysines.  Using BLAST algorithms we found CBU_1932 had no similarity with currently defined proteins, but has orthologues in other human intracellular pathogens such as Legionella and Chlamydia. Due to the high number of basic residues in CBU_1932, and linkage with a hypothetical DNA binding protein (CBU_1933), we hypothesize that CBU_1932 may also encode a protein involved with binding DNA or other negatively charged substrates.  To address this hypothesis, we are in the process of cloning the 201-base pair CBU_1932 ORF into pMAL-c5x expression plasmid and analyzing the recombinant protein using DNA-binding protocols including electrophoretic mobility-shift assay EMSA.  We are confident that characterization of this high-level transcript/highly basic protein will lead to a better understanding of the unique metabolism of Coxiella and other intracellular pathogens

Nivel de adherencia al tratamiento y calidad de vida relacionada a la salud en pacientes con VIH/sida, de un hospital MINSA nivel II-1 de Ferreñafe – Lambayeque, durante agosto – diciembre, 2017

Se ha observado que pacientes infectados por el virus de inmunodeficiencia humana y síndrome de inmunodeficiencia adquirida (VIH/SIDA) que no toman en cuenta las indicaciones del médico para su tratamiento, pueden generar una propagación constante y continua, dando paso a un problema de salud más grave a nivel mundial. Por ello se realizó una investigación no experimental - descriptiva, con el propósito de determinar los niveles de adherencia al tratamiento y calidad de vida relacionada a la salud según sexo y grado de instrucción, además de describir las dimensiones sociodemográficas en pacientes con VIH/SIDA, de un Hospital MINSA nivel II-1 de Ferreñafe - Lambayeque, durante agosto a diciembre del 2017. Se utilizó los cuestionarios CEAT-VIH para hallar los niveles de adherencia al tratamiento y MOS-SF 30 para determinar los niveles de calidad de vida relacionada a la salud, estimando su validez y confiabilidad en una población similar, siendo válidos y fiables. Además, en cuanto la variable sexo, se obtuvo que los varones tienen una adecuada adherencia al tratamiento, sin embargo para calidad de vida relacionada a la salud ambos sexos se encontraron en un nivel bajo. Por ende la población estudiada presenta baja adherencia al tratamiento y calidad de vida relacionada a la salud. Con respecto a los aspectos sociodemográficos, el 53% es de sexo masculino, el 62% es soltera, siendo la vía sexual (96%) la principal forma de transmisión del virus del VIH. Además el 75% lleva un tiempo mayor a 12 meses de tratamiento en el servicio de TARGA.Tesi

Identification of Novel Small RNAs and Characterization of the 6S RNA of \u3ci\u3eCoxiella burnetii\u3c/i\u3e

Coxiella burnetii, an obligate intracellular bacterial pathogen that causes Q fever, undergoes a biphasic developmental cycle that alternates between a metabolically-active large cell variant (LCV) and a dormant small cell variant (SCV). As such, the bacterium undoubtedly employs complex modes of regulating its lifecycle, metabolism and pathogenesis. Small RNAs (sRNAs) have been shown to play important regulatory roles in controlling metabolism and virulence in several pathogenic bacteria. We hypothesize that sRNAs are involved in regulating growth and development of C. burnetii and its infection of host cells. To address the hypothesis and identify potential sRNAs, we subjected total RNA isolated from Coxiella cultured axenically and in Vero host cells to deep-sequencing. Using this approach, we identified fifteen novel C. burnetii sRNAs (CbSRs). Fourteen CbSRs were validated by Northern blotting. Most CbSRs showed differential expression, with increased levels in LCVs. Eight CbSRs were upregulated ($2-fold) during intracellular growth as compared to growth in axenic medium. Along with the fifteen sRNAs, we also identified three sRNAs that have been previously described from other bacteria, including RNase P RNA, tmRNA and 6S RNA. The 6S regulatory sRNA of C. burnetii was found to accumulate over log phase-growth with a maximum level attained in the SCV stage. The 6S RNA-encoding gene (ssrS) was mapped to the 59 UTR of ygfA; a highly conserved linkage in eubacteria. The predicted secondary structure of the 6S RNA possesses three highly conserved domains found in 6S RNAs of other eubacteria. We also demonstrate that Coxiella’s 6S RNA interacts with RNA polymerase (RNAP) in a specific manner. Finally, transcript levels of 6S RNA were found to be at much higher levels when Coxiella was grown in host cells relative to axenic culture, indicating a potential role in regulating the bacterium’s intracellular stress response by interacting with RNAP during transcription

Characterization and expression analysis of the groESL operon of Bartonella bacilliformis

The groESL operon of Bartonella bacilliformis, a facultative intracellular, Gram-negative bacterium and etiologic agent of Oroya Fever, was characterized. Sequence analysis revealed an operon containing two genes of 294 (groES) and 1632 nucleotides (groEL) separated by a 55-nt intergenic spacer. The operon is preceded by a 72-nt ORF (ORF1) that encodes a hypothetical protein with homology to a portion of the HrcA repressor for groESL. A divergent fumarate hydratase C (fumC) gene lies further upstream. Deduced amino acid sequences for B. bacilliformis GroEL and GroES revealed a high degree of identity with homologues from other Bartonella and alpha-Protebacteria. A single transcriptional start site (TSS) was mapped 79 nucleotides upstream of the groES start codon, regardless of incubation temperature. The TSS was located immediately 5\u27 to a potential controlling inverted repeat of chaperonin expression (CIRCE) element and is preceded by a sigma70-like promoter. The operon is followed by a predicted rho-independent transcriptional terminator. Northern blot analysis indicated that groES and groEL are co-transcribed as a single mRNA of approximately 2.4 kb. A 6-h time course analysis by qRT-PCR showed that groEL expression increases 1.3-fold within 30 min of a temperature upshift from 30 to 37 degrees C, with maximum transcription reached after 60 min (approximately 4.3-fold), followed by a steady decrease to background (30 degrees C) transcription levels by 6 h. Western blot analysis revealed a 1.4- and 1.5-fold increase in GroEL synthesis following a temperature upshift or by inhibiting DNA supercoiling with coumermycin A1, respectively. Functional expression and complementation of temperature-sensitive Escherichia coli groES or groEL mutants with the cloned operon allowed them to grow at otherwise restrictive temperatures