Mobile genetic elements in Coxiella burnetii: Friends, foes or just indifferent?

The genome of the obligate intracellular pathogen Coxiella burnetii contains a large number of mobile genetic elements including two group I introns and an intervening sequence (IVS) that interrupt the 23S rRNA gene; an intein within dnaB (encoding replicative DNA helicase) and a homing endonuclease. The introns are self-splicing ribozymes and able to inhibit ribosome function and retard bacterial growth through internal guide sequence (IGS)-dependent and -independent mechanisms. The introns were found to be highly conserved in all eight genomic groups of C. burnetii, suggesting a role in C. burnetii\u27s biology. It is not clear whether the introns are being positively selected because they promote bacterial persistence or whether they are slightly deleterious but were fixed in the population due to genetic drift. The intein is able to self-splice, leaving the host protein intact and presumably functional. Similar inteins were found in two extremophilic bacteria (Alkalilimnicola ehrlichei and Halorhodospira halophila) that were found to be distantly related to Coxiella. Intein splicing appears to be a slow process, making it possible that before the intein is excised the DnaB precursor is non-functional, thereby reducing the pool of mature DnaB, thus creating a lag in replication and slowing growth. The IVS is found inserted into helix 45 of C. burnetii\u27s 23S rRNA. Unlike introns and inteins, the flanking regions are not spliced back together after the IVS is excised, resulting in a fragmented 23S rRNA. The IVS encodes a hypothetical protein that is conserved between IVSs in a wide variety of bacteria. Phylogenetic analyses revealed that a similar ORF is present on an IVS inserted at the same locus in many Leptospira species, suggesting horizontal gene transfer as the mode of spread of this genetic element. It is possible that the dramatic drop in ribosome quantities that occurs when Coxiella transitions from LCV to SCV is aided by RNA fragmentation caused by the IVS. Another mobile genetic element found in C. burnetii is a homing endonuclease encoded within an intron. Here, we have characterized four selfish genetic elements in C. burnetii and show that they share an intimate relationship with their host

Accumulation and Expression of Multiple Antibiotic Resistance Genes in \u3ci\u3eArcobacter Cryaerophilus\u3c/i\u3e that Thrives in Sewage

We explored the bacterial diversity of untreated sewage influent samples of a wastewater treatment plant in Tucson, AZ and discovered that Arcobacter cryaerophilus, an emerging human pathogen of animal origin, was the most dominant bacterium. The other highly prevalent bacteria were members of the phyla Bacteroidetes and Firmicutes, which are major constituents of human gut microbiome, indicating that bacteria of human and animal origin intermingle in sewage. By assembling a near-complete genome of A. cryaerophilus, we show that the bacterium has accumulated a large number of antibiotic resistance genes (ARGs) probably enabling it to thrive in the wastewater. We also determined that a majority of ARGs was being expressed in sewage, suggestive of trace levels of antibiotics or other stresses that could act as a selective force that amplifies multidrug resistant bacteria in municipal sewage. Because all bacteria are not eliminated even after several rounds of wastewater treatment, ARGs in sewage could affect public health due to their potential to contaminate environmental water

Genetics of Coxiella burnetii: on the path of specialization

Coxiella burnetii is an extremely infectious, zoonotic agent that causes Q fever in humans. With the exception of New Zealand, the bacterium is distributed worldwide. Coxiella is classified as a select agent based on its past and potential use as a bioweapon and its threat to public health. Despite decades of research, we know relatively little regarding Coxiella?s molecular pathogenesis, and a vaccine is not widely available. This article briefly reviews the unusual genetics of C. burnetii; a pathogen that retains telltale genetic mementos collected over the course of its evolutionary path from a free-living bacterium to an obligate intracellular parasite of eukaryotic host cell phagosomes. Understanding why these genetic elements are maintained may help us better understand the biology of this fascinating pathogen

A Selective Force Favoring Increased G+C Content in Bacterial Genes

Bacteria display considerable variation in their overall base compositions, which range from 13% to over 75% G+C. This variation in genomic base compositions has long been considered to be a strictly neutral character, due solely to differences in the mutational process; however, recent sequence comparisons indicate that mutational input alone cannot produce the observed base compositions, implying a role for natural selection. Because bacterial genomes have high gene content, forces that operate on the base composition of individual genes could help shape the overall genomic base composition. To explore this possibility, we tested whether genes that encode the same protein but vary only in their base compositions at synonymous sites have effects on bacterial fitness. Escherichia coli strains harboring G+C-rich versions of genes display higher growth rates, indicating that despite a pervasive mutational bias toward A+T, a selective force, independent of adaptive codon use, is driving genes toward higher G+C contents

A Unique Group I Intron in Coxiella Burnetii is a Natural Splice Mutant

Cbu.L1917, a group I intron present in the 23S rRNA gene of Coxiella burnetii, possesses a unique 3\u27-terminal adenine in place of a conserved guanine. Here, we show that, unlike all other group I introns, Cbu.L1917 utilizes a different cofactor for each splicing step and has a decreased self-splicing rate in vitro

MicroRNAs Contribute to the Host Response to Coxiella burnetii

MicroRNAs (miRNAs), a class of small non-coding RNAs, are critical to gene regulation in eukaryotes. They are involved in modulating a variety of physiological processes, including the host response to intracellular infections. Little is known about miRNA functions during infection by Coxiella burnetii, the causative agent of human Q fever. This bacterial pathogen establishes a large replicative vacuole within macrophages by manipulating host processes such as apoptosis and autophagy. We investigated miRNA expression in C. burnetii-infected macrophages and identified several miRNAs that were down- or up-regulated during infection. We further explored the functions of miR-143-3p, an miRNA whose expression is down-regulated in macrophages infected with C. burnetii, and show that increasing the abundance of this miRNA in human cells results in increased apoptosis and reduced autophagy – conditions that are unfavorable to C. burnetii intracellular growth. In sum, this study demonstrates that C. burnetii infection elicits a robust miRNA-based host response, and because miR-143-3p promotes apoptosis and inhibits autophagy, down-regulation of miR-143-3p expression during C. burnetii infection likely benefits the pathogen

Partnership between diverse stakeholders: A potential solution to issues migrant construction workers face in Bengaluru, India

While partnerships between diverse stakeholders could improve the lives of migrant construction workers (MCW) in Bengaluru, literature on partnerships is limited. The purpose of this dissertation is to: i) document the perspectives on the response to issues that MCW face, ii) identify opportunities for improving the response through partnerships. Guided by a theoretical framework I developed, I collected qualitative data using focus groups (n=3) and interviews (n=2) with female MCW/family members in small construction sites, informal settlements, and a company site; interviews with representatives of civil society organizations (CSO) (n=6), the construction sector (n=10) and the government (n=6); and participant observation in Bengaluru for eight months. I analyzed the data using a combination of predetermined and emergent themes and sub-themes. I worked with members of a community advisory board throughout the dissertation. I found MCW move to Bengaluru for job opportunities and better wages. In Bengaluru, MCW face substandard working and living conditions and limited access to services and resources, which affect women more adversely. While CSO, the construction sector, and the government have taken initiatives to improve MCW’ lives, their reach is limited with differences based on the setting. Partnerships, existing and potential, address access to services, skill development, infrastructure creation, and registration with social protection programs. Partnerships within stakeholders and those involving multiple stakeholders can increase partnership effectiveness. However, partnerships are not suited to address MCW’ rights and the needs of MCW in small construction sites. Participants did not volunteer solutions to issues female MCW/family members face. Funding, trust, wariness about CSO, slow government decision-making process, and fear of bureaucracy affect the formation and functioning of existing and potential partnerships. There is an opportunity to improve the existing response to issues MCW face through partnerships but with limitations. To overcome these limitations, empowering MCW is crucial. This study’s significance stems from completing stakeholder scoping and identifying issues that partnerships can address, which is the first step in establishing partnerships. Future research needs to explore further the factors that impact the functioning of partnerships and ways of mitigating them along with identifying mechanisms for upholding MCW’ rights

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

Impact of endosymbionts on tick physiology and fitness

Ticks transmit pathogens and harbour non-pathogenic, vertically transmitted intracellular bacteria termed endosymbionts. Almost all ticks studied to date contain 1 or more of Coxiella, Francisella, Rickettsia or Candidatus Midichloria mitochondrii endosymbionts, indicative of their importance to tick physiology. Genomic and experimental data suggest that endosymbionts promote tick development and reproductive success. Here, we review the limited information currently available on the potential roles endosymbionts play in enhancing tick metabolism and fitness. Future studies that expand on these findings are needed to better understand endosymbionts’ contributions to tick biology. This knowledge could potentially be applied to design novel strategies that target endosymbiont function to control the spread of ticks and pathogens they vector