Publications by authors named "Clayton C Caswell"

40 Publications

Enemy of My Enemy: A Novel Insect-Specific Flavivirus Offers a Promising Platform for a Zika Virus Vaccine.

Vaccines (Basel) 2021 Oct 7;9(10). Epub 2021 Oct 7.

Department of Entomology, Fralin Life Science Institute, Virginia Tech, Blacksburg, VA 24061, USA.

Vaccination remains critical for viral disease outbreak prevention and control, but conventional vaccine development typically involves trade-offs between safety and immunogenicity. We used a recently discovered insect-specific flavivirus as a vector in order to develop an exceptionally safe, flavivirus vaccine candidate with single-dose efficacy. To evaluate the safety and efficacy of this platform, we created a chimeric Zika virus (ZIKV) vaccine candidate, designated Aripo/Zika virus (ARPV/ZIKV). ZIKV has caused immense economic and public health impacts throughout the Americas and remains a significant public health threat. ARPV/ZIKV vaccination showed exceptional safety due to ARPV/ZIKV's inherent vertebrate host-restriction. ARPV/ZIKV showed no evidence of replication or translation in vitro and showed no hematological, histological or pathogenic effects in vivo. A single-dose immunization with ARPV/ZIKV induced rapid and robust neutralizing antibody and cellular responses, which offered complete protection against ZIKV-induced morbidity, mortality and in utero transmission in immune-competent and -compromised murine models. Splenocytes derived from vaccinated mice demonstrated significant CD4 and CD8 responses and significant cytokine production post-antigen exposure. Altogether, our results further support that chimeric insect-specific flaviviruses are a promising strategy to restrict flavivirus emergence via vaccine development.
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http://dx.doi.org/10.3390/vaccines9101142DOI Listing
October 2021

Presumptive Identification of Smooth Strain Antibodies in Canines.

Front Vet Sci 2021 8;8:697479. Epub 2021 Jul 8.

Department of Small Animal Clinical Sciences, Virginia-Maryland College of Veterinary Medicine, , Blacksburg, VA, United States.

Brucellosis is a zoonotic disease caused by a Gram-negative coccobacillus. There are four strains of zoonotic importance in our domestic species, subdivided by their culture phenotypes: (), (smooth strains) and (rough strain). Dogs can serve as hosts for all four of the zoonotic strains; however, routine serologic testing in dogs has been limited to the identification of antibodies. The aim of our study was to identify smooth strain antibodies in canines. We hypothesize that the Fluorescence Polarization Assay would be successful in identifying smooth Brucella strain antibodies in canines. Ninety-five dogs, including forty-five hog hunting dogs were screened for circulating antibodies to any of the four zoonotic strains of the bacteria utilizing a combination of Canine Slide Agglutination Test (CBSA), Agar Gel Immunodiffusion II test (AGIDII), Card Agglutination Test (BCA), and the Fluorescence Polarization Assay (FPA). Test interpretation results yielded a 0% (0/95) smooth strain seropositivity rate, with 2% (2/95) of dogs yielding inconclusive rough strain serology results (0-2% rough strain seropositivity rate). Additionally, a retrospective portion of the study was performed to identify sera containing circulating antibodies to any of the smooth strains of by testing previously banked canine serum samples stored at Cornell's Veterinary Diagnostic Laboratory from 2018 to 2019 via FPA. Of the 769 serum samples tested, 13/769 (1.7%) yielded an inconclusive result, 725/769 (94.2%) were negative, 30/769 (4%) yielded a positive FPA test result, and 1/769 (0.1%) had to be excluded due to insufficient sample remaining to perform the diagnostic test. Of the 30 FPA positive canine serum samples, 97% (29/30) also tested positive on the CBSA test. Additionally, there was a statistically significant ( < 0.0001) likelihood of altered (spayed/neutered) and mixed breed dogs to be FPA positive when compared to intact, purebred dogs, respectively.
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http://dx.doi.org/10.3389/fvets.2021.697479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8295921PMC
July 2021

ASC-Mediated Inflammation and Pyroptosis Attenuates Pathogenesis Following the Recognition of gDNA.

Pathogens 2020 Nov 30;9(12). Epub 2020 Nov 30.

Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24061, USA.

is a zoonotic pathogen that causes brucellosis. Because of unique LPS layer and intracellular localization predominately within macrophages, it can often evade immune detection. However, pattern recognition receptors are capable of sensing pathogen-associated molecular patterns (PAMPS). For example, NOD-like receptors (NLRs) can form a multi-protein inflammasome complex to attenuate pathogenesis. The inflammasome activates IL-1β and IL-18 to drive immune cell recruitment. Alternatively, inflammasome activation also initiates inflammatory cell death, termed pyroptosis, which augments bacteria clearance. In this report, we assess canonical and non-canonical inflammasome activation following infection. We conducted in vivo studies using mice and observed decreased mouse survival, immune cell recruitment, and increased bacteria load. We also conducted studies with mice and did not observe any significant impact on pathogenesis. Through mechanistic studies using macrophages, our data suggests that the protective role of ASC may result from the induction of pyroptosis through a gasdermin D-dependent mechanism in macrophages. Additionally, we show that the recognition of is facilitated by sensing the PAMP gDNA rather than the less immunogenic LPS. Together, these results refine our understanding of the role that inflammasome activation and pyroptosis plays during brucellosis.
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http://dx.doi.org/10.3390/pathogens9121008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7760712PMC
November 2020

A central role for the transcriptional regulator VtlR in small RNA-mediated gene regulation in Agrobacterium tumefaciens.

Sci Rep 2020 09 11;10(1):14968. Epub 2020 Sep 11.

Center for One Health Research, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, 24060, USA.

LysR-type transcriptional regulators (LTTRs) are the most common type of transcriptional regulators in prokaryotes and function by altering gene expression in response to environmental stimuli. In the class Alphaproteobacteria, a conserved LTTR named VtlR is critical to the establishment of host-microbe interactions. In the mammalian pathogen Brucella abortus, VtlR is required for full virulence in a mouse model of infection, and VtlR activates the expression of abcR2, which encodes a small regulatory RNA (sRNA). In the plant symbiont Sinorhizobium meliloti, the ortholog of VtlR, named LsrB, is involved in the symbiosis of the bacterium with alfalfa. Agrobacterium tumefaciens is a close relative of both B. abortus and S. meliloti, and this bacterium is the causative agent of crown gall disease in plants. In the present study, we demonstrate that VtlR is involved in the ability of A. tumefaciens to grow appropriately in artificial medium, and an A. tumefaciens vtlR deletion strain is defective in motility, biofilm formation, and tumorigenesis of potato discs. RNA-sequencing analyses revealed that more than 250 genes are dysregulated in the ∆vtlR strain, and importantly, VtlR directly controls the expression of three sRNAs in A. tumefaciens. Taken together, these data support a model in which VtlR indirectly regulates hundreds of genes via manipulation of sRNA pathways in A. tumefaciens, and moreover, while the VtlR/LsrB protein is present and structurally conserved in many members of the Alphaproteobacteria, the VtlR/LsrB regulatory circuitry has diverged in order to accommodate the unique environmental niche of each organism.
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http://dx.doi.org/10.1038/s41598-020-72117-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7486931PMC
September 2020

Characterizing the transport and utilization of the neurotransmitter GABA in the bacterial pathogen Brucella abortus.

PLoS One 2020 26;15(8):e0237371. Epub 2020 Aug 26.

Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Blacksburg, Virginia, United States of America.

The neurotransmitter gamma-aminobutyric acid (GABA) is the most abundant inhibitory neurotransmitter in the human brain; however, it is becoming more evident that this non-proteinogenic amino acid plays multiple physiological roles in biology. In the present study, the transport and function of GABA is studied in the highly infectious intracellular bacterium Brucella abortus. The data show that 3H-GABA is imported by B. abortus under nutrient limiting conditions and that the small RNAs AbcR1 and AbcR2 negatively regulate this transport. A specific transport system, gts, is responsible for the transport of GABA as determined by measuring 3H-GABA transport in isogenic deletion strains of known AbcR1/2 regulatory targets; however, this locus is unnecessary for Brucella infection in BALB/c mice. Similar assays revealed that 3H-GABA transport is uninhibited by the 20 standard proteinogenic amino acids, representing preference for the transport of 3H-GABA. Metabolic studies did not show any potential metabolic utilization of GABA by B. abortus as a carbon or nitrogen source, and RNA sequencing analysis revealed limited transcriptional differences between B. abortus 2308 with or without exposure to GABA. While this study provides evidence for GABA transport by B. abortus, questions remain as to why and when this transport is utilized during Brucella pathogenesis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0237371PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7449393PMC
October 2020

The Endoribonuclease RNase E Coordinates Expression of mRNAs and Small Regulatory RNAs and Is Critical for the Virulence of Brucella abortus.

J Bacteriol 2020 09 23;202(20). Epub 2020 Sep 23.

Center for One Health Research, Virginia-Maryland College of Veterinary Medicine, Blacksburg, Virginia, USA

RNases are key regulatory components in prokaryotes, responsible for the degradation and maturation of specific RNA molecules at precise times. Specifically, RNases allow cells to cope with changes in their environment through rapid alteration of gene expression. To date, few RNases have been characterized in the mammalian pathogen In the present work, we sought to investigate several RNases in and determine what role, if any, they have in pathogenesis. Of the 4 RNases reported in this study, the highly conserved endoribonuclease, RNase E, was found to play an integral role in the virulence of Although , which encodes RNase E, is essential in , we were able to generate a strain encoding a defective version of RNase E lacking the C-terminal portion of the protein, and this strain (-tnc) was attenuated in a mouse model of infection. RNA-sequencing analysis revealed massive RNA dysregulation in -tnc, with 122 upregulated and 161 downregulated transcripts compared to the parental strain. Interestingly, several mRNAs related to metal homeostasis were significantly decreased in the -tnc strain. We also identified a small regulatory RNA (sRNA), called Bsr4, that exhibited significantly elevated levels in -tnc, demonstrating an important role for RNase E in sRNA-mediated regulatory pathways in Overall, these data highlight the importance of RNase E in , including the role of RNase E in properly controlling mRNA levels and contributing to virulence in an animal model of infection. Brucellosis is a debilitating disease of humans and animals globally, and there is currently no vaccine to combat human infection by spp. Moreover, effective antibiotic treatment in humans is extremely difficult and can lead to disease relapse. Therefore, it is imperative that systems and pathways be identified and characterized in the brucellae so new vaccines and therapies can be generated. In this study, we describe the impact of the endoribonuclease RNase E on the control of mRNA and small regulatory RNA (sRNA) levels in , as well as the importance of RNase E for the full virulence of This work greatly enhances our understanding of ribonucleases in the biology and pathogenesis of spp.
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http://dx.doi.org/10.1128/JB.00240-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7515240PMC
September 2020

Sinorhizobium meliloti YbeY is a zinc-dependent single-strand specific endoribonuclease that plays an important role in 16S ribosomal RNA processing.

Nucleic Acids Res 2020 01;48(1):332-348

Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.

Single-strand specific endoribonuclease YbeY has been shown to play an important role in the processing of the 3' end of the 16S rRNA in Escherichia coli. Lack of YbeY results in the accumulation of the 17S rRNA precursor. In contrast to a previous report, we show that Sinorhizobium meliloti YbeY exhibits endoribonuclease activity on single-stranded RNA substrate but not on the double-stranded substrate. This study also identifies the previously unknown metal ion involved in YbeY function to be Zn2+ and shows that the activity of YbeY is enhanced when the occupancy of zinc is increased. We have identified a pre-16S rRNA precursor that accumulates in the S. meliloti ΔybeY strain. We also show that ΔybeY mutant of Brucella abortus, a mammalian pathogen, also accumulates a similar pre-16S rRNA. The pre-16S species is longer in alpha-proteobacteria than in gamma-proteobacteria. We demonstrate that the YbeY from E. coli and S. meliloti can reciprocally complement the rRNA processing defect in a ΔybeY mutant of the other organism. These results establish YbeY as a zinc-dependent single-strand specific endoribonuclease that functions in 16S rRNA processing in both alpha- and gamma-proteobacteria.
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http://dx.doi.org/10.1093/nar/gkz1095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6943124PMC
January 2020

Quantitative Variation in m.3243A > G Mutation Produce Discrete Changes in Energy Metabolism.

Sci Rep 2019 04 8;9(1):5752. Epub 2019 Apr 8.

Fralin Biomedical Research Institute at Virginia Tech Carilion, Roanoke, VA, 24016, USA.

Mitochondrial DNA (mtDNA) 3243A > G tRNALeu heteroplasmic mutation (m.3243A > G) exhibits clinically heterogeneous phenotypes. While the high mtDNA heteroplasmy exceeding a critical threshold causes mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes (MELAS) syndrome, the low mtDNA heteroplasmy causes maternally inherited diabetes with or without deafness (MIDD) syndrome. How quantitative differences in mtDNA heteroplasmy produces distinct pathological states has remained elusive. Here we show that despite striking similarities in the energy metabolic gene expression signature, the mitochondrial bioenergetics, biogenesis and fuel catabolic functions are distinct in cells harboring low or high levels of the m.3243 A > G mutation compared to wild type cells. We further demonstrate that the low heteroplasmic mutant cells exhibit a coordinate induction of transcriptional regulators of the mitochondrial biogenesis, glucose and fatty acid metabolism pathways that lack in near homoplasmic mutant cells compared to wild type cells. Altogether, these results shed new biological insights on the potential mechanisms by which low mtDNA heteroplasmy may progressively cause diabetes mellitus.
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http://dx.doi.org/10.1038/s41598-019-42262-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6453956PMC
April 2019

Assessment of Survival and Replication of Brucella spp. in Murine Peritoneal Macrophages.

Methods Mol Biol 2019 ;1960:181-189

Department of Biomedical Sciences and Pathobiology, Center for One Health Research, Virginia Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, USA.

Brucella spp. are bacteria that naturally infect a variety of domesticated and wild animals leading to abortions and infertility, and these bacteria are also capable of causing debilitating human infections, which often result from human exposure to infected animals and animal products. The brucellae are intracellular pathogens that reside in host cells, including macrophages and dendritic cells, and it is paramount for the pathogenesis of Brucella that the bacteria are able to survive and replicate in these host cells. The methods outlined in this chapter can be employed to study the interactions between Brucella strains and primary murine peritoneal macrophages.
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http://dx.doi.org/10.1007/978-1-4939-9167-9_16DOI Listing
July 2019

Defining the regulatory mechanism of NikR, a nickel-responsive transcriptional regulator, in Brucella abortus.

Microbiology (Reading) 2018 10 31;164(10):1320-1325. Epub 2018 Jul 31.

Department of Biomedical Sciences and Pathobiology, Center for One Health Research, VA-MD College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24060, USA.

Metals are essential micronutrients for virtually all forms of life, but metal acquisition is a double-edged sword, because high concentrations of divalent cations can be toxic to the cell. Therefore, the genes involved in metal acquisition, storage and efflux are tightly regulated. The present study characterizes a nickel-responsive transcriptional regulator in the intracellular mammalian pathogen, Brucella abortus. Deletion of bab2_0432 (nikR) in B. abortus led to alterations in the nickel-responsive expression of the genes encoding the putative nickel importer NikABCDE and, moreover, NikR binds directly to a specific DNA sequence within the promoter region of nikA in a metal-dependent manner to control gene expression. While NikR is involved in controlling the expression of nikA, nikR is not required for the infection of macrophages or mice by B. abortus. Overall, this work characterizes the role of NikR in nickel-responsive gene expression, as well as the dispensability of nikR for Brucella virulence.
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http://dx.doi.org/10.1099/mic.0.000702DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6600342PMC
October 2018

Characterization of Three Small Proteins in Brucella abortus Linked to Fucose Utilization.

J Bacteriol 2018 09 24;200(18). Epub 2018 Aug 24.

Department of Biomedical Sciences and Pathobiology, Center for One Health Research, VA-MD College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia, USA

Elucidating the function of proteins <50 amino acids in length is no small task. Nevertheless, small proteins can play vital roles in the lifestyle of bacteria and influence the virulence of pathogens; thus, the investigation of the small proteome is warranted. Recently, our group identified the protein VtlR as a transcriptional activator of four genes, one of which is the well-studied small regulatory RNA AbcR2, while the other three genes encode hypothetical small proteins, two of which are highly conserved among the order This study provides evidence that all three genes encode authentic small proteins and that all three are highly expressed under oxidative stress, low-pH, and stationary-phase growth conditions. Fractionation of the cells revealed that the proteins are localized to the membranes of We demonstrate that the small proteins under the transcriptional control of VtlR are not accountable for attenuation observed with the deletion strain. However, there is an association between VtlR-regulated genes and growth inhibition in the presence of the sugar l-fucose. Subsequent transcriptomic analyses revealed that initiates the transcription of a locus encoding a putative sugar transport and utilization system when the bacteria are cultured in the presence of l-fucose. Altogether, our observations characterize the role of the VtlR-controlled small proteins BAB1_0914, BAB2_0512, and BAB2_0574 in the biology of , particularly in the capacity of the bacteria to utilize l-fucose. Despite being one of the most common zoonoses worldwide, there is currently no human vaccine to combat brucellosis. Therefore, a better understanding of the pathogenesis and biology of spp., the causative agent of brucellosis, is essential for the discovery of novel therapeutics against these highly infectious bacteria. In this study, we further characterize the virulence-associated transcriptional regulator VtlR in Our findings not only shed light on our current understanding of a virulence related genetic system in spp. but also increase our knowledge of small proteins in the field of bacteriology.
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http://dx.doi.org/10.1128/JB.00127-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6112010PMC
September 2018

Endoribonuclease YbeY Is Linked to Proper Cellular Morphology and Virulence in Brucella abortus.

J Bacteriol 2018 06 24;200(12). Epub 2018 May 24.

Department of Biomedical Sciences and Pathobiology, VA-MD College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia, USA

The YbeY endoribonuclease is one of the best-conserved proteins across the kingdoms of life. In the present study, we demonstrated that YbeY in is linked to a variety of important activities, including proper cellular morphology, mRNA transcript levels, and virulence. Deletion of in led to a small-colony phenotype when the bacteria were grown on agar medium, as well as to significant aberrations in the morphology of the bacterial cell as evidenced by electron microscopy. Additionally, compared to the parental strain, the Δ strain was significantly attenuated in both macrophage and mouse models of infection. The Δ strain also showed increased sensitivities to several -applied stressors, including bile acid, hydrogen peroxide, SDS, and paraquat. Transcriptomic analysis revealed that a multitude of mRNA transcripts are dysregulated in the Δ strain, and many of the identified mRNAs encode proteins involved in metabolism, nutrient transport, transcriptional regulation, and flagellum synthesis. We subsequently constructed gene deletion strains of the most highly dysregulated systems, and several of the YbeY-linked gene deletion strains exhibited defects in the ability of the bacteria to survive and replicate in primary murine macrophages. Taken together, these data establish a clear role for YbeY in the biology and virulence of ; moreover, this work further illuminates the highly varied roles of this widely conserved endoribonuclease in bacteria. spp. are highly efficient bacterial pathogens of animals and humans, causing significant morbidity and economic loss worldwide, and relapse of disease often occurs following antibiotic treatment of human brucellosis. As such, novel therapeutic strategies to combat infections are needed. Ribonucleases in the brucellae are understudied, and these enzymes represent elements that may be potential targets for future treatment approaches. The present work demonstrates the importance of the YbeY endoribonuclease for cellular morphology, efficient control of mRNA levels, and virulence in Overall, the results of this study advance our understanding of the critical roles of YbeY in the pathogenesis of the intracellular brucellae and expand our understanding of this highly conserved RNase.
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http://dx.doi.org/10.1128/JB.00105-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5971472PMC
June 2018

An account of evolutionary specialization: the AbcR small RNAs in the Rhizobiales.

Mol Microbiol 2018 Jan 17;107(1):24-33. Epub 2017 Nov 17.

Department of Biomedical Sciences and Pathobiology, VA-MD College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, USA.

The AbcR small RNAs (sRNAs) are a fascinating example of two highly conserved sRNAs that differ tremendously at the functional level among organisms. From their transcriptional activation to their regulatory capabilities, the AbcR sRNAs exhibit varying characteristics in three well-studied bacteria belonging to the Rhizobiales order: the plant symbiont Sinorhizobium meliloti, the plant pathogen Agrobacterium tumefaciens, and the animal pathogen Brucella abortus. This review outlines the similarities and differences of the AbcR sRNAs between each of these organisms, and discusses reasons as to why this group of sRNAs has diverged in their genetic organization and regulatory functions across species. In the end, this review will shed light on how regulatory systems, although seemingly conserved among bacteria, can vary based on the environmental niche and lifestyle of an organism.
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http://dx.doi.org/10.1111/mmi.13869DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5752129PMC
January 2018

Proline utilization system is required for infection by the pathogenic α-proteobacterium Brucella abortus.

Microbiology (Reading) 2017 07 21;163(7):970-979. Epub 2017 Jul 21.

Department of Biomedical Sciences and Pathobiology, Center for Molecular Medicine and Infectious Diseases, VA-MD College of Veterinary Medicine, Virginia Tech, Blacksburg, VA 24060, USA.

Proline utilization (Put) systems have been described in a number of bacteria; however, the importance and functionality of the Put system in the intracellular pathogen Brucellaabortus has not been explored. Generally, bacterial Put systems are composed of the bifunctional enzyme proline dehydrogenase PutA and its transcriptional activator PutR. Here, we demonstrate that the genes putA (bab2_0518) and putR (bab2_0517) are critical for the chronic infection of mice by B. abortus, but putA and putR are not required for the survival and replication of the bacteria in naive macrophages. Additionally, in vitro experiments revealed that putR is necessary for the ability of the bacteria to withstand oxidative stress, as a ΔputR deletion strain is hypersensitive to hydrogen peroxide exposure. Quantitative reverse transcription-PCR and putA-lacZ transcriptional reporter studies revealed that PutR acts as a transcriptional activator of putA in Brucella, and electrophoretic mobility shift assays confirmed that PutR binds directly to the putA promoter region. Biochemical analyses demonstrated that a purified recombinant B. abortus PutA protein possesses quintessential proline dehydrogenase activity, as PutA is capable of catalysing the conversion of proline to glutamate. Altogether, these data are the first to reveal that the Put system plays a significant role in the ability of B. abortus to replicate and survive within its host, as well as to describe the genetic regulation and biochemical activity of the Put system in Brucella.
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http://dx.doi.org/10.1099/mic.0.000490DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737144PMC
July 2017

A 6-Nucleotide Regulatory Motif within the AbcR Small RNAs of Mediates Host-Pathogen Interactions.

mBio 2017 06 6;8(3). Epub 2017 Jun 6.

Department of Biomedical Sciences and Pathobiology, Center for Molecular Medicine and Infectious Diseases, VA-MD College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia, USA

In , two small RNAs (sRNAs), AbcR1 and AbcR2, are responsible for regulating transcripts encoding ABC-type transport systems. AbcR1 and AbcR2 are required for virulence, as a double chromosomal deletion of both sRNAs results in attenuation in mice. Although these sRNAs are responsible for targeting transcripts for degradation, the mechanism utilized by the AbcR sRNAs to regulate mRNA in has not been described. Here, two motifs (M1 and M2) were identified in AbcR1 and AbcR2, and complementary motif sequences were defined in AbcR-regulated transcripts. Site-directed mutagenesis of M1 or M2 or of both M1 and M2 in the sRNAs revealed transcripts to be targeted by one or both motifs. Electrophoretic mobility shift assays revealed direct, concentration-dependent binding of both AbcR sRNAs to a target mRNA sequence. These experiments genetically and biochemically characterized two indispensable motifs within the AbcR sRNAs that bind to and regulate transcripts. Additionally, cellular and animal models of infection demonstrated that only M2 in the AbcR sRNAs is required for virulence. Furthermore, one of the M2-regulated targets, BAB2_0612, was found to be critical for the virulence of in a mouse model of infection. Although these sRNAs are highly conserved among , the present report displays how gene regulation mediated by the AbcR sRNAs has diverged to meet the intricate regulatory requirements of each particular organism and its unique biological niche. Small RNAs (sRNAs) are important components of bacterial regulation, allowing organisms to quickly adapt to changes in their environments. The AbcR sRNAs are highly conserved throughout the and negatively regulate myriad transcripts, many encoding ABC-type transport systems. In , AbcR1 and AbcR2 are functionally redundant, as only a double (/) deletion results in attenuation and In the present study, we confirmed that the AbcR sRNAs have redundant regulatory functions and defined two six-nucleotide motifs, M1 and M2, that the AbcR sRNAs utilize to control gene expression. Importantly, only M2 was linked to virulence. Further investigation of M2-regulated targets identified BAB2_0612 as critical for colonization of in mice, highlighting the significance of AbcR M2-regulated transcripts for infection. Overall, our findings define the molecular mechanism of the virulence-associated AbcR system in the pathogenic bacterium .
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http://dx.doi.org/10.1128/mBio.00473-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5461406PMC
June 2017

Sibling sRNA RyfA1 Influences Shigella dysenteriae Pathogenesis.

Genes (Basel) 2017 Jan 26;8(2). Epub 2017 Jan 26.

Department of Biomedical Sciences, Ohio University Heritage College of Osteopathic Medicine, 1 Ohio University Drive Athens, Athens, OH 45701, USA.

Small regulatory RNAs (sRNAs) of Shigella dysenteriae and other pathogens are vital for the regulation of virulence-associated genes and processes. Here, we characterize RyfA1, one member of a sibling pair of sRNAs produced by S. dysenteriae. Unlike its nearly identical sibling molecule, RyfA2, predicted to be encoded almost exclusively by non-pathogenic species, the presence of a gene encoding RyfA1, or a RyfA1-like molecule, is strongly correlated with virulence in a variety of enteropathogens. In S. dysenteriae, the overproduction of RyfA1 negatively impacts the virulence-associated process of cell-to-cell spread as well as the expression of ompC, a gene encoding a major outer membrane protein important for the pathogenesis of Shigella. Interestingly, the production of RyfA1 is controlled by a second sRNA, here termed RyfB1, the first incidence of one regulatory small RNA controlling another in S. dysenteriae or any Shigella species.
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http://dx.doi.org/10.3390/genes8020050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5333039PMC
January 2017

Enhanced Mucosal Defense and Reduced Tumor Burden in Mice with the Compromised Negative Regulator IRAK-M.

EBioMedicine 2017 Feb 3;15:36-47. Epub 2016 Dec 3.

Department of Biomedical Sciences and Pathobiology, VA-MD College of Veterinary Medicine, Blacksburg, Virginia 24061, United States. Electronic address:

Aberrant inflammation is a hallmark of inflammatory bowel disease (IBD) and colorectal cancer. IRAK-M is a critical negative regulator of TLR signaling and overzealous inflammation. Here we utilize data from human studies and Irak-m mice to elucidate the role of IRAK-M in the modulation of gastrointestinal immune system homeostasis. In human patients, IRAK-M expression is up-regulated during IBD and colorectal cancer. Further functional studies in mice revealed that Irak-m animals are protected against colitis and colitis associated tumorigenesis. Mechanistically, our data revealed that the gastrointestinal immune system of Irak-m mice is highly efficient at eliminating microbial translocation following epithelial barrier damage. This attenuation of pathogenesis is associated with expanded areas of gastrointestinal associated lymphoid tissue (GALT), increased neutrophil migration, and enhanced T-cell recruitment. Further evaluation of Irak-m mice revealed a splice variant that robustly activates NF-κB signaling. Together, these data identify IRAK-M as a potential target for future therapeutic intervention.
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http://dx.doi.org/10.1016/j.ebiom.2016.11.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5233813PMC
February 2017

Strain 2308 Wisconsin Genome: Importance of the Definition of Reference Strains.

Front Microbiol 2016 29;7:1557. Epub 2016 Sep 29.

Programa de Investigación en Enfermedades Tropicales, Escuela de Medicina Veterinaria, Universidad Nacional de Costa RicaHeredia, Costa Rica; Centro de Investigación en Enfermedades Tropicales, Facultad de Microbiología, Universidad de Costa RicaSan José, Costa Rica.

Brucellosis is a bacterial infectious disease affecting a wide range of mammals and a neglected zoonosis caused by species of the genetically homogenous genus . As in most studies on bacterial diseases, research in brucellosis is carried out by using reference strains as canonical models to understand the mechanisms underlying host pathogen interactions. We performed whole genome sequencing analysis of the reference strain 2308 routinely used in our laboratory, including manual curated annotation accessible as an editable version through a link at https://en.wikipedia.org/wiki/Brucella#Genomics. Comparison of this genome with two publically available 2308 genomes showed significant differences, particularly indels related to insertional elements, suggesting variability related to the transposition of these elements within the same strain. Considering the outcome of high resolution genomic techniques in the bacteriology field, the conventional concept of strain definition needs to be revised.
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http://dx.doi.org/10.3389/fmicb.2016.01557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5041503PMC
September 2016

Unique Footprint in the scl1.3 Locus Affects Adhesion and Biofilm Formation of the Invasive M3-Type Group A Streptococcus.

Front Cell Infect Microbiol 2016 31;6:90. Epub 2016 Aug 31.

Department of Microbiology, Immunology, and Cell Biology, West Virginia University Morgantown, WV, USA.

The streptococcal collagen-like proteins 1 and 2 (Scl1 and Scl2) are major surface adhesins that are ubiquitous among group A Streptococcus (GAS). Invasive M3-type strains, however, have evolved two unique conserved features in the scl1 locus: (i) an IS1548 element insertion in the scl1 promoter region and (ii) a nonsense mutation within the scl1 coding sequence. The scl1 transcript is drastically reduced in M3-type GAS, contrasting with a high transcription level of scl1 allele in invasive M1-type GAS. This leads to a lack of Scl1 expression in M3 strains. In contrast, while scl2 transcription and Scl2 production are elevated in M3 strains, M1 GAS lack Scl2 surface expression. M3-type strains were shown to have reduced biofilm formation on inanimate surfaces coated with cellular fibronectin and laminin, and in human skin equivalents. Repair of the nonsense mutation and restoration of Scl1 expression on M3-GAS cells, restores biofilm formation on cellular fibronectin and laminin coatings. Inactivation of scl1 in biofilm-capable M28 and M41 strains results in larger skin lesions in a mouse model, indicating that lack of Scl1 adhesin promotes bacterial spread over localized infection. These studies suggest the uniquely evolved scl1 locus in the M3-type strains, which prevents surface expression of the major Scl1 adhesin, contributed to the emergence of the invasive M3-type strains. Furthermore these studies provide insight into the molecular mechanisms mediating colonization, biofilm formation, and pathogenesis of group A streptococci.
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http://dx.doi.org/10.3389/fcimb.2016.00090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5005324PMC
September 2017

Transcriptome-Wide Identification of Hfq-Associated RNAs in Brucella suis by Deep Sequencing.

J Bacteriol 2016 02 9;198(3):427-35. Epub 2015 Nov 9.

INSERM U1047, Nîmes, France Université de Montpellier, UFR Médecine, Nîmes, France

Unlabelled: Recent breakthroughs in next-generation sequencing technologies have led to the identification of small noncoding RNAs (sRNAs) as a new important class of regulatory molecules. In prokaryotes, sRNAs are often bound to the chaperone protein Hfq, which allows them to interact with their partner mRNA(s). We screened the genome of the zoonotic and human pathogen Brucella suis 1330 for the presence of this class of RNAs. We designed a coimmunoprecipitation strategy that relies on the use of Hfq as a bait to enrich the sample with sRNAs and eventually their target mRNAs. By deep sequencing analysis of the Hfq-bound transcripts, we identified a number of mRNAs and 33 sRNA candidates associated with Hfq. The expression of 10 sRNAs in the early stationary growth phase was experimentally confirmed by Northern blotting and/or reverse transcriptase PCR.

Importance: Brucella organisms are facultative intracellular pathogens that use stealth strategies to avoid host defenses. Adaptation to the host environment requires tight control of gene expression. Recently, small noncoding RNAs (sRNAs) and the sRNA chaperone Hfq have been shown to play a role in the fine-tuning of gene expression. Here we have used RNA sequencing to identify RNAs associated with the B. suis Hfq protein. We have identified a novel list of 33 sRNAs and 62 Hfq-associated mRNAs for future studies aiming to understand the intracellular lifestyle of this pathogen.
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http://dx.doi.org/10.1128/JB.00711-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4719455PMC
February 2016

A LysR-family transcriptional regulator required for virulence in Brucella abortus is highly conserved among the α-proteobacteria.

Mol Microbiol 2015 Oct 14;98(2):318-28. Epub 2015 Aug 14.

Department of Biomedical Sciences and Pathobiology, Center for Molecular Medicine and Infectious Diseases, VA-MD College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, 24060, USA.

Small RNAs are principal elements of bacterial gene regulation and physiology. Two small RNAs in Brucella abortus, AbcR1 and AbcR2, are required for wild-type virulence. Examination of the abcR loci revealed the presence of a gene encoding a LysR-type transcriptional regulator flanking abcR2 on chromosome 1. Deletion of this lysR gene (bab1_1517) resulted in the complete loss of abcR2 expression while no difference in abcR1 expression was observed. The B. abortus bab1_1517 mutant strain was significantly attenuated in macrophages and mice, and bab1_1517 was subsequently named vtlR for virulence-associated transcriptional LysR-family regulator. Microarray analysis revealed three additional genes encoding small hypothetical proteins also under the control of VtlR. Electrophoretic mobility shift assays demonstrated that VtlR binds directly to the promoter regions of abcR2 and the three hypothetical protein-encoding genes, and DNase I footprint analysis identified the specific nucleotide sequence in these promoters that VtlR binds to and drives gene expression. Strikingly, orthologs of VtlR are encoded in a wide range of host-associated α-proteobacteria, and it is likely that the VtlR genetic system represents a common regulatory circuit critical for host-bacterium interactions.
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http://dx.doi.org/10.1111/mmi.13123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5846693PMC
October 2015

Coordinated zinc homeostasis is essential for the wild-type virulence of Brucella abortus.

J Bacteriol 2015 May 17;197(9):1582-91. Epub 2015 Feb 17.

Department of Biomedical Sciences and Pathobiology, Center for Molecular Medicine and Infectious Diseases, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, Blacksburg, Virginia, USA

Unlabelled: Metal homeostasis in bacterial cells is a highly regulated process requiring intricately coordinated import and export, as well as precise sensing of intracellular metal concentrations. The uptake of zinc (Zn) has been linked to the virulence of Brucella abortus; however, the capacity of Brucella strains to sense Zn levels and subsequently coordinate Zn homeostasis has not been described. Here, we show that expression of the genes encoding the zinc uptake system ZnuABC is negatively regulated by the Zn-sensing Fur family transcriptional regulator, Zur, by direct interactions between Zur and the promoter region of znuABC. Moreover, the MerR-type regulator, ZntR, controls the expression of the gene encoding the Zn exporter ZntA by binding directly to its promoter. Deletion of zur or zntR alone did not result in increased zinc toxicity in the corresponding mutants; however, deletion of zntA led to increased sensitivity to Zn but not to other metals, such as Cu and Ni, suggesting that ZntA is a Zn-specific exporter. Strikingly, deletion of zntR resulted in significant attenuation of B. abortus in a mouse model of chronic infection, and subsequent experiments revealed that overexpression of zntA in the zntR mutant is the molecular basis for its decreased virulence.

Importance: The importance of zinc uptake for Brucella pathogenesis has been demonstrated previously, but to date, there has been no description of how overall zinc homeostasis is maintained and genetically controlled in the brucellae. The present work defines the predominant zinc export system, as well as the key genetic regulators of both zinc uptake and export in Brucella abortus. Moreover, the data show the importance of precise coordination of the zinc homeostasis systems as disregulation of some elements of these systems leads to the attenuation of Brucella virulence in a mouse model. Overall, this study advances our understanding of the essential role of zinc in the pathogenesis of intracellular bacteria.
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http://dx.doi.org/10.1128/JB.02543-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4403653PMC
May 2015

Sibling rivalry: related bacterial small RNAs and their redundant and non-redundant roles.

Front Cell Infect Microbiol 2014 28;4:151. Epub 2014 Oct 28.

Department of Biomedical Sciences, Ohio University Heritage College of Osteopathic Medicine Athens, OH, USA.

Small RNA molecules (sRNAs) are now recognized as key regulators controlling bacterial gene expression, as sRNAs provide a quick and efficient means of positively or negatively altering the expression of specific genes. To date, numerous sRNAs have been identified and characterized in a myriad of bacterial species, but more recently, a theme in bacterial sRNAs has emerged: the presence of more than one highly related sRNAs produced by a given bacterium, here termed sibling sRNAs. Sibling sRNAs are those that are highly similar at the nucleotide level, and while it might be expected that sibling sRNAs exert identical regulatory functions on the expression of target genes based on their high degree of relatedness, emerging evidence is demonstrating that this is not always the case. Indeed, there are several examples of bacterial sibling sRNAs with non-redundant regulatory functions, but there are also instances of apparent regulatory redundancy between sibling sRNAs. This review provides a comprehensive overview of the current knowledge of bacterial sibling sRNAs, and also discusses important questions about the significance and evolutionary implications of this emerging class of regulators.
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http://dx.doi.org/10.3389/fcimb.2014.00151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4211561PMC
January 2015

Bacterial persistence: finding the "sweet spot".

Cell Host Microbe 2013 Aug;14(2):119-20

Department of Microbiology and Immunology, Brody School of Medicine, East Carolina University, Greenville, NC 27858, USA.

Studies described by Eisele et al. (2013) and Xavier et al. (2013) in this issue of Cell Host & Microbe show that the bacterial pathogens Salmonella and Brucella exploit the increased levels of glucose present in alternatively activated macrophages to sustain chronic infections in experimentally infected mice.
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http://dx.doi.org/10.1016/j.chom.2013.07.016DOI Listing
August 2013

The Brucella abortus general stress response system regulates chronic mammalian infection and is controlled by phosphorylation and proteolysis.

J Biol Chem 2013 May 1;288(19):13906-16. Epub 2013 Apr 1.

Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, IL 60637, USA.

Background: Virulence of pathogenic bacteria is often determined by their ability to adapt to stress.

Results: The Brucella abortus general stress response (GSR) system is required for chronic mammalian infection and is regulated by phosphorylation and proteolysis.

Conclusion: The B. abortus GSR signaling pathway has multiple layers of post-translational control and is a determinant of chronic infection.

Significance: This study provides new, molecular level insight into chronic Brucella infection. Brucella spp. are adept at establishing a chronic infection in mammals. We demonstrate that core components of the α-proteobacterial general stress response (GSR) system, PhyR and σ(E1), are required for Brucella abortus stress survival in vitro and maintenance of chronic murine infection in vivo. ΔphyR and ΔrpoE1 null mutants exhibit decreased survival under acute oxidative and acid stress but are not defective in infection of primary murine macrophages or in initial colonization of BALB/c mouse spleens. However, ΔphyR and ΔrpoE1 mutants are attenuated in spleens beginning 1 month postinfection. Thus, the B. abortus GSR system is dispensable for colonization but is required to maintain chronic infection. A genome-scale analysis of the B. abortus GSR regulon identified stress response genes previously linked to virulence and genes that affect immunomodulatory components of the cell envelope. These data support a model in which the GSR system affects both stress survival and the interface between B. abortus and the host immune system. We further demonstrate that PhyR proteolysis is a unique feature of GSR control in B. abortus. Proteolysis of PhyR provides a mechanism to avoid spurious PhyR protein interactions that inappropriately activate GSR-dependent transcription. We conclude that the B. abortus GSR system regulates acute stress adaptation and long term survival within a mammalian host and that PhyR proteolysis is a novel regulatory feature in B. abortus that ensures proper control of GSR transcription.
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http://dx.doi.org/10.1074/jbc.M113.459305DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3650426PMC
May 2013

Diverse genetic regulon of the virulence-associated transcriptional regulator MucR in Brucella abortus 2308.

Infect Immun 2013 Apr 14;81(4):1040-51. Epub 2013 Jan 14.

Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, North Carolina, USA.

The Ros-type regulator MucR is one of the few transcriptional regulators that have been linked to virulence in Brucella. Here, we show that a Brucella abortus in-frame mucR deletion strain exhibits a pronounced growth defect during in vitro cultivation and, more importantly, that the mucR mutant is attenuated in cultured macrophages and in mice. The genetic basis for the attenuation of Brucella mucR mutants has not been defined previously, but in the present study the genes regulated by MucR in B. abortus have been elucidated using microarray analysis and real-time reverse transcription-PCR (RT-PCR). In B. abortus 2308, MucR regulates a wide variety of genes whose products may function in establishing and maintaining cell envelope integrity, polysaccharide biosynthesis, iron homeostasis, genome plasticity, and transcriptional regulation. Particularly notable among the MucR-regulated genes identified is arsR6 (nolR), which encodes a transcriptional regulator previously linked to virulence in Brucella melitensis 16 M. Importantly, electrophoretic mobility shift assays (EMSAs) determined that a recombinant MucR protein binds directly to the promoter regions of several genes repressed by MucR (including arsR6 [nolR]), and in Brucella, as in other alphaproteobacteria, MucR binds to its own promoter to repress expression of the gene that encodes it. Overall, these studies have uncovered the diverse genetic regulon of MucR in Brucella, and in doing so this work has begun to define the MucR-controlled genetic circuitry whose misregulation contributes to the virulence defect of Brucella mucR mutants.
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http://dx.doi.org/10.1128/IAI.01097-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3639602PMC
April 2013

Characterization of the organic hydroperoxide resistance system of Brucella abortus 2308.

J Bacteriol 2012 Sep 20;194(18):5065-72. Epub 2012 Jul 20.

Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, North Carolina, USA.

The organic hydroperoxide resistance protein Ohr has been identified in numerous bacteria where it functions in the detoxification of organic hydroperoxides, and expression of ohr is often regulated by a MarR-type regulator called OhrR. The genes annotated as BAB2_0350 and BAB2_0351 in the Brucella abortus 2308 genome sequence are predicted to encode OhrR and Ohr orthologs, respectively. Using isogenic ohr and ohrR mutants and lacZ promoter fusions, it was determined that Ohr contributes to resistance to organic hydroperoxide, but not hydrogen peroxide, in B. abortus 2308 and that OhrR represses the transcription of both ohr and ohrR in this strain. Moreover, electrophoretic mobility shift assays and DNase I footprinting revealed that OhrR binds directly to a specific region in the intergenic region between ohr and ohrR that shares extensive nucleotide sequence similarity with so-called "OhrR boxes" described in other bacteria. While Ohr plays a prominent role in protecting B. abortus 2308 from organic hydroperoxide stress in in vitro assays, this protein is not required for the wild-type virulence of this strain in cultured murine macrophages or experimentally infected mice.
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http://dx.doi.org/10.1128/JB.00873-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3430311PMC
September 2012

Identification of two small regulatory RNAs linked to virulence in Brucella abortus 2308.

Mol Microbiol 2012 Jul 12;85(2):345-60. Epub 2012 Jun 12.

Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, NC 27834, USA.

Hfq is an RNA-binding protein that functions in post-transcriptional gene regulation by mediating interactions between mRNAs and small regulatory RNAs (sRNAs). Two proteins encoded by BAB1_1794 and BAB2_0612 are highly over-produced in a Brucella abortus hfq mutant compared with the parental strain, and recently, expression of orthologues of these proteins in Agrobacterium tumefaciens was shown to be regulated by two sRNAs, called AbcR1 and AbcR2. Orthologous sRNAs (likewise designated AbcR1 and AbcR2) have been identified in B. abortus 2308. In Brucella, abcR1 and abcR2 single mutants are not defective in their ability to survive in cultured murine macrophages, but an abcR1 abcR2 double mutant exhibits significant attenuation in macrophages. Additionally, the abcR1 abcR2 double mutant displays significant attenuation in a mouse model of chronic Brucella infection. Quantitative proteomics and microarray analyses revealed that the AbcR sRNAs predominantly regulate genes predicted to be involved in amino acid and polyamine transport and metabolism, and Northern blot analyses indicate that the AbcR sRNAs accelerate the degradation of the target mRNAs. In an Escherichia coli two-plasmid reporter system, overexpression of either AbcR1 or AbcR2 was sufficient for regulation of target mRNAs, indicating that the AbcR sRNAs from B. abortus 2308 perform redundant regulatory functions.
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http://dx.doi.org/10.1111/j.1365-2958.2012.08117.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3391331PMC
July 2012

Redox-responsive regulation of denitrification genes in Brucella.

Mol Microbiol 2012 Jul 30;85(1):5-7. Epub 2012 May 30.

Department of Microbiology and Immunology, Brody School of Medicine, East Carolina University, Greenville, NC 27834, USA.

Brucella strains encounter oxygen deprivation during their intracellular replication in host cells, and the capacity of these bacteria to utilize NO(3) as an alternative electron acceptor for respiration plays an important role in their successful adaption to their intracellular niche. In this issue of Molecular Microbiology, Carrica et al (2012). report that NtrY and NtrX comprise a redox-responsive two-component regulator in Brucella abortus 2308 that responds to decreasing levels of O(2) and induces the expression of this strain's denitrification genes. Thus, NtrYX joins the increasing number of genetic regulators that contribute to the metabolic versatility required for the virulence of Brucella strains in their mammalian hosts.
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http://dx.doi.org/10.1111/j.1365-2958.2012.08096.xDOI Listing
July 2012

Mur regulates the gene encoding the manganese transporter MntH in Brucella abortus 2308.

J Bacteriol 2012 Feb 18;194(3):561-6. Epub 2011 Nov 18.

Department of Microbiology and Immunology, Brody School of Medicine, East Carolina University, Greenville, North Carolina, USA.

MntH is the only high-affinity manganese transporter identified in Brucella. A previous study showed that MntH is required for the wild-type virulence of Brucella abortus 2308 in mice (Anderson ES, et al., Infect. Immun. 77:3466-3474, 2009) and indicated that the mntH gene is regulated in a manganese-responsive manner in this strain by a Mur homolog. In the study presented here, the transcriptional start site for mntH in B. abortus 2308 was determined by primer extension analysis. Specific interactions between Mur and the mntH promoter region were demonstrated in an electrophoretic mobility shift assay (EMSA), and a Mur binding site was identified in the -55 to -24 region of the mntH promoter by DNase I footprint analysis. The specificity of the interaction of Mur with the putative Mur box was further evaluated by EMSA employing oligonucleotides in which the consensus nucleotides in this region were substituted. These studies not only confirm a direct role for Mur in the Mn-responsive regulation of mntH expression in Brucella abortus 2308 but also identify the cis-acting elements upstream of mntH that are responsible for this regulation.
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http://dx.doi.org/10.1128/JB.05296-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3264066PMC
February 2012
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