scholarly journals The Borrelia burgdorferi Adenylate Cyclase, CyaB, Is Important for Virulence Factor Production and Mammalian Infection

2021 ◽  
Vol 12 ◽  
Author(s):  
Vanessa M. Ante ◽  
Lauren C. Farris ◽  
Elizabeth P. Saputra ◽  
Allie J. Hall ◽  
Nathaniel S. O’Bier ◽  
...  
Keyword(s):  
Gene Expression ◽  
Borrelia Burgdorferi ◽  
Adenylate Cyclase ◽  
Direct Detection ◽  
Adenosine Monophosphate ◽  
Host Cells ◽  
Mammalian Host ◽  
Environmental Signals ◽  
Mammalian Cell Lines ◽  
Mammalian Infection

Borrelia burgdorferi, the causative agent of Lyme disease, traverses through vastly distinct environments between the tick vector and the multiple phases of the mammalian infection that requires genetic adaptation for the progression of pathogenesis. Borrelial gene expression is highly responsive to changes in specific environmental signals that initiate the RpoS regulon for mammalian adaptation, but the mechanism(s) for direct detection of environmental cues has yet to be identified. Secondary messenger cyclic adenosine monophosphate (cAMP) produced by adenylate cyclase is responsive to environmental signals, such as carbon source and pH, in many bacterial pathogens to promote virulence by altering gene regulation. B. burgdorferi encodes a single non-toxin class IV adenylate cyclase (bb0723, cyaB). This study investigates cyaB expression along with its influence on borrelial virulence regulation and mammalian infectivity. Expression of cyaB was specifically induced with co-incubation of mammalian host cells that was not observed with cultivated tick cells suggesting that cyaB expression is influenced by cellular factor(s) unique to mammalian cell lines. The 3′ end of cyaB also encodes a small RNA, SR0623, in the same orientation that overlaps with bb0722. The differential processing of cyaB and SR0623 transcripts may alter the ability to influence function in the form of virulence determinant regulation and infectivity. Two independent cyaB deletion B31 strains were generated in 5A4-NP1 and ML23 backgrounds and complemented with the cyaB ORF alone that truncates SR0623, cyaB with intact SR0623, or cyaB with a mutagenized full-length SR0623 to evaluate the influence on transcriptional and posttranscriptional regulation of borrelial virulence factors and infectivity. In the absence of cyaB, the expression and production of ospC was significantly reduced, while the protein levels for BosR and DbpA were substantially lower than parental strains. Infectivity studies with both independent cyaB mutants demonstrated an attenuated phenotype with reduced colonization of tissues during early disseminated infection. This work suggests that B. burgdorferi utilizes cyaB and potentially cAMP as a regulatory pathway to modulate borrelial gene expression and protein production to promote borrelial virulence and dissemination in the mammalian host.

scholarly journals The Borrelia burgdorferi adenylyl cyclase, CyaB, is important for virulence factor production and mammalian infection

2021 ◽  
Author(s):  
Vanessa M Ante ◽  
Lauren C Farris ◽  
Elizabeth P Saputra ◽  
Allie J Hall ◽  
Nathaniel S O'Bier ◽  
...  
Keyword(s):  
Gene Expression ◽  
Borrelia Burgdorferi ◽  
Adenylate Cyclase ◽  
Direct Detection ◽  
Adenosine Monophosphate ◽  
Host Cells ◽  
Mammalian Host ◽  
Environmental Signals ◽  
Mammalian Cell Lines ◽  
Mammalian Infection

Borrelia burgdorferi, the causative agent of Lyme disease, traverses through vastly distinct environments between the tick vector and the multiple phases of the mammalian infection that requires genetic adaptation for the progression of pathogenesis. Borrelial gene expression is highly responsive to changes in specific environmental signals that initiate the RpoS regulon for mammalian adaptation, but the mechanism(s) for direct detection of environmental cues has yet to be identified. Secondary messenger cyclic adenosine monophosphate (cAMP) produced by adenylate cyclase is responsive to environmental signals, such as carbon source and pH, in many bacterial pathogens to promote virulence by altering gene regulation. B. burgdorferi encodes a single non-toxin class IV adenylate cyclase (bb0723, cyaB). This study investigates cyaB expression along with its influence on borrelial virulence regulation and mammalian infectivity. Expression of cyaB was specifically induced with co-incubation of mammalian host cells that was not observed with cultivated tick cells suggesting that cyaB expression is influenced by cellular factor(s) unique to mammalian cell lines. The 3' end of cyaB also encodes a small RNA, SR0623, in the same orientation that overlaps with bb0722. The differential processing of cyaB and SR0623 transcripts may alter the ability to influence function in the form of virulence determinant regulation and infectivity. Two independent cyaB deletion B31 strains were generated in 5A4-NP1 and ML23 backgrounds and complemented with the cyaB ORF alone that truncates SR0623, cyaB with intact SR0623, or cyaB with a mutagenized full length SR0623 to evaluate the influence on transcriptional and post-transcriptional regulation of borrelial virulence factors and infectivity. In the absence of cyaB, expression and production of ospC was significantly reduced, while the protein levels for BosR and DbpA were substantially lower than parental strains. Infectivity studies with both independent cyaB mutants demonstrated an attenuated phenotype with reduced colonization of tissues during early disseminated infection. This work suggests that B. burgdorferi utilizes cyaB and potentially cAMP as a regulatory pathway to modulate borrelial gene expression and protein production to promote borrelial virulence and dissemination in the mammalian host.

scholarly journals Role of the BBA64 Locus of Borrelia burgdorferi in Early Stages of Infectivity in a Murine Model of Lyme Disease

2007 ◽  
Vol 76 (1) ◽  
pp. 391-402 ◽  
Author(s):  
Mahulena Maruskova ◽  
M. Dolores Esteve-Gassent ◽  
Valerie L. Sexton ◽  
J. Seshu
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Immunoblot Analysis ◽  
Open Reading Frames ◽  
Mammalian Host ◽  
Environmental Signals ◽  
Significant Difference ◽  
Linear Plasmid 54

ABSTRACT Borrelia burgdorferi, the causative agent of Lyme disease, undergoes rapid adaptive gene expression in response to environmental signals encountered during different stages of its life cycle in the arthropod vector or the mammalian host. Among all the plasmid-encoded genes of B. burgdorferi, several linear plasmid 54 (lp54)-encoded open reading frames (ORFs) exhibit the greatest differential expression in response to mammalian host-specific temperature, pH, and other uncharacterized signals. These ORFs include members of the paralogous gene family 54 (pgf 54), such as BBA64, BBA65, and BBA66, present on lp54. In an attempt to correlate transcriptional up-regulation of these pgf 54 members to their role in infectivity, we inactivated BBA64 and characterized the phenotype of this mutant both in vitro and in vivo. There were no major differences in the protein profiles between the BBA64 mutant and the control strains, while immunoblot analysis indicated that inactivation of BBA64 resulted in increased levels of BBA65. Moreover, there was no significant difference in the ability of the BBA64 mutant to infect C3H/HeN mice compared to that of its parental or complemented control strains as determined by culturing of viable spirochetes from infected tissues. However, enumeration of spirochetes using quantitative real-time PCR revealed tissue-specific differences, suggesting a minimal role for BBA64 in the survival of B. burgdorferi in select tissues. Infectivity analysis of the BBA64 mutant suggests that B. burgdorferi may utilize multiple determinants to establish infection in mammalian hosts.

An enhanced GFP reporter system to monitor gene expression in Borrelia burgdorferi

Microbiology ◽  
2003 ◽  
Vol 149 (7) ◽  
pp. 1819-1828 ◽  
Author(s):  
James A. Carroll ◽  
Philip E. Stewart ◽  
Patricia Rosa ◽  
Abdallah F. Elias ◽  
Claude F. Garon
Keyword(s):  
Gene Expression ◽  
Borrelia Burgdorferi ◽  
Fluorescent Protein ◽  
Regulation Of Gene Expression ◽  
Epifluorescence Microscopy ◽  
Reporter System ◽  
Environmental Signals ◽  
Gfp Reporter ◽  
Green Fluorescent

Borrelia burgdorferi regulates genes in response to a number of environmental signals such as temperature and pH. A green fluorescent protein (GFP) reporter system using the ospC, ospA and flaB promoters from B. burgdorferi B31 was introduced into infectious clonal isolates of strains B31 and N40 to monitor and compare gene expression in response to pH and temperature in vitro. GFP could be assayed by epifluorescence microscopy, immunoblotting or spectrofluorometry and was an accurate reporter of target gene expression. It was determined that only 179 bp 5′ of ospC was sufficient to regulate the reporter gfp in vitro in response to pH and temperature in B. burgdorferi B31. The loss of linear plasmid (lp) 25, lp28-1, lp36 and lp56 had no effect on the ability of B. burgdorferi B31 to regulate ospC in response to pH or temperature. The amount of OspC in N40 transformants was unaffected by changes in pH or temperature of the culture medium. This suggests that regulation of gene expression in response to pH and temperature may vary between these two B. burgdorferi strains.

scholarly journals Borrelia burgdorferi Alters Its Gene Expression and Antigenic Profile in Response to CO2 Levels

2006 ◽  
Vol 189 (2) ◽  
pp. 437-445 ◽  
Author(s):  
Jenny A. Hyde ◽  
Jerome P. Trzeciakowski ◽  
Jonathan T. Skare
Keyword(s):  
Gene Expression ◽  
Protein Synthesis ◽  
Borrelia Burgdorferi ◽  
Growth Conditions ◽  
Etiologic Agent ◽  
Complex Life Cycle ◽  
Mammalian Host ◽  
Transcriptional Level ◽  
Virulence Determinants

ABSTRACT The etiologic agent of Lyme disease, Borrelia burgdorferi, must adapt to the distinct environments of its arthropod vector and mammalian host during its complex life cycle. B. burgdorferi alters gene expression and protein synthesis in response to temperature, pH, and other uncharacterized environmental factors. The hypothesis tested in this study is that dissolved gases, including CO2, serve as a signal for B. burgdorferi to alter protein production and gene expression. In this study we focused on characterization of in vitro anaerobic (5% CO2, 3% H2, 0.087 ppm O2) and microaerophilic (1% CO2, 3.48 ppm O2) growth conditions and how they modulate protein synthesis and gene expression in B. burgdorferi. Higher levels of several immunoreactive proteins, including BosR, NapA, DbpA, OspC, BBK32, and RpoS, were synthesized under anaerobic conditions. Previous studies demonstrated that lower levels of NapA were produced when microaerophilic cultures were purged with nitrogen gas to displace oxygen and CO2. In this study we identified CO2 as a factor contributing to the observed change in NapA synthesis. Specifically, a reduction in the level of dissolved CO2, independent of O2 levels, resulted in reduced NapA synthesis. BosR, DbpA, OspC, and RpoS synthesis was also decreased with the displacement of CO2. Quantitative reverse transcription-PCR indicated that the levels of the dbpA, ospC, and BBK32 transcripts are increased in the presence of CO2, indicating that these putative borrelial virulence determinants are regulated at the transcriptional level. Thus, dissolved CO2 may be an additional cue for borrelial host adaptation and gene regulation.

scholarly journals Dissolved Oxygen Levels Alter Gene Expression and Antigen Profiles in Borrelia burgdorferi

2004 ◽  
Vol 72 (3) ◽  
pp. 1580-1586 ◽  
Author(s):  
J. Seshu ◽  
Julie A. Boylan ◽  
Frank C. Gherardini ◽  
Jonathan T. Skare
Keyword(s):  
Gene Expression ◽  
Borrelia Burgdorferi ◽  
Dissolved Oxygen ◽  
Reverse Transcription ◽  
Host Factors ◽  
Pcr Analysis ◽  
Transcriptional Level ◽  
Environmental Signals ◽  
Reverse Transcription Pcr ◽  
Genes Encoding

ABSTRACT The Lyme disease spirochete, Borrelia burgdorferi, encounters many environmental signals as it cycles between the arthropod vector and mammalian hosts, including temperature, pH, and other host factors. To test the possibility that dissolved oxygen modulates gene expression in B. burgdorferi, spirochetes were exposed to differential levels of dissolved oxygen, and distinct alterations were observed at both the transcriptional and translational levels. Specifically NapA, a Dps/Dpr homologue involved in the oxidative stress response in other bacteria, was reduced when B. burgdorferi was grown under oxygen-limiting conditions. In contrast, several immunoreactive proteins were altered when tested with infection-derived sera from different hosts. Specifically, OspC, DbpA, and VlsE were synthesized at greater levels when cells were grown in limiting oxygen, whereas VraA was reduced. The levels of oxygen in the medium did not affect OspA production. Real-time reverse transcription-PCR analysis of RNA isolated from infectious isolates of strains B31 and cN40 indicated that the expression of ospC, dbpA, and vlsE increased while napA expression decreased under dissolved-oxygen-limiting conditions, whereas flaB was not affected. The reverse transcription-PCR results corroborated the immunoblot analyses and indicated that the increase in OspC, DbpA, and VlsE was due to regulation at the transcriptional level of the genes encoding these antigens. These results indicate that dissolved oxygen modulates gene expression in B. burgdorferi and imply that the redox environment may be an additional regulatory cue that spirochetes exploit to adapt to the disparate niches that they occupy in nature.

scholarly journals Gene Expression Changes Induced byTrypanosoma cruziShed Microvesicles in Mammalian Host Cells: Relevance of tRNA-Derived Halves

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Maria R. Garcia-Silva ◽  
Florencia Cabrera-Cabrera ◽  
Roberta Ferreira Cura das Neves ◽  
Thaís Souto-Padrón ◽  
Wanderley de Souza ◽  
...  
Keyword(s):  
Gene Expression ◽  
Host Cell ◽  
Extracellular Vesicles ◽  
Small Rna ◽  
Hela Cells ◽  
Small Rnas ◽  
Host Cells ◽  
Mammalian Host ◽  
Large Set ◽  
Rna Pathways

At present, noncoding small RNAs are recognized as key players in novel forms of posttranscriptional gene regulation in most eukaryotes. However, canonical small RNA pathways seem to be lost or excessively simplified in some unicellular organisms includingTrypanosoma cruziwhich lack functional RNAi pathways. Recently, we reported the presence of alternate small RNA pathways inT. cruzimainly represented by homogeneous populations of tRNA- and rRNA-derived small RNAs, which are secreted to the extracellular medium included in extracellular vesicles. Extracellular vesicle cargo could be delivered to other parasites and to mammalian susceptible cells promoting metacyclogenesis and conferring susceptibility to infection, respectively. Here we analyzed the changes in gene expression of host HeLa cells induced by extracellular vesicles fromT. cruzi. As assessed by microarray assays a large set of genes in HeLa cells were differentially expressed upon incorporation ofT. cruzi-derived extracellular vesicles. The elicited response modified mainly host cell cytoskeleton, extracellular matrix, and immune responses pathways. Some genes were also modified by the most abundant tRNA-derived small RNAs included in extracellular vesicles. These data suggest that microvesicles secreted byT. cruzicould be relevant players in early events of theT. cruzihost cell interplay.

scholarly journals Mapping the Regulatory Network for Salmonella enterica Serovar Typhimurium Invasion

mBio ◽  
2016 ◽  
Vol 7 (5) ◽  
Author(s):  
Carol Smith ◽  
Anne M. Stringer ◽  
Chunhong Mao ◽  
Michael J. Palumbo ◽  
Joseph T. Wade
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Cross Talk ◽  
Regulatory Network ◽  
Salmonella Enterica ◽  
Expression Profiles ◽  
Pathogenicity Island ◽  
Host Cells ◽  
Environmental Signals ◽  
Regulatory Pathways

ABSTRACT Salmonella enterica pathogenicity island 1 (SPI-1) encodes proteins required for invasion of gut epithelial cells. The timing of invasion is tightly controlled by a complex regulatory network. The transcription factor (TF) HilD is the master regulator of this process and senses environmental signals associated with invasion. HilD activates transcription of genes within and outside SPI-1, including six other TFs. Thus, the transcriptional program associated with host cell invasion is controlled by at least 7 TFs. However, very few of the regulatory targets are known for these TFs, and the extent of the regulatory network is unclear. In this study, we used complementary genomic approaches to map the direct regulatory targets of all 7 TFs. Our data reveal a highly complex and interconnected network that includes many previously undescribed regulatory targets. Moreover, the network extends well beyond the 7 TFs, due to the inclusion of many additional TFs and noncoding RNAs. By comparing gene expression profiles of regulatory targets for the 7 TFs, we identified many uncharacterized genes that are likely to play direct roles in invasion. We also uncovered cross talk between SPI-1 regulation and other regulatory pathways, which, in turn, identified gene clusters that likely share related functions. Our data are freely available through an intuitive online browser and represent a valuable resource for the bacterial research community. IMPORTANCE Invasion of epithelial cells is an early step during infection by Salmonella enterica and requires secretion of specific proteins into host cells via a type III secretion system (T3SS). Most T3SS-associated proteins required for invasion are encoded in a horizontally acquired genomic locus known as Salmonella pathogenicity island 1 (SPI-1). Multiple regulators respond to environmental signals to ensure appropriate timing of SPI-1 gene expression. In particular, there are seven transcription regulators that are known to be involved in coordinating expression of SPI-1 genes. We have used complementary genome-scale approaches to map the gene targets of these seven regulators. Our data reveal a highly complex and interconnected regulatory network that includes many previously undescribed target genes. Moreover, our data functionally implicate many uncharacterized genes in the invasion process and reveal cross talk between SPI-1 regulation and other regulatory pathways. All datasets are freely available through an intuitive online browser.

scholarly journals Global Analysis of Borrelia burgdorferi Genes Regulated by Mammalian Host-Specific Signals

2003 ◽  
Vol 71 (6) ◽  
pp. 3371-3383 ◽  
Author(s):  
Chad S. Brooks ◽  
P. Scott Hefty ◽  
Sarah E. Jolliff ◽  
Darrin R. Akins
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Differentially Expressed Genes ◽  
Global Analysis ◽  
Outer Membrane Proteins ◽  
Differentially Expressed ◽  
Mammalian Host ◽  
Global Changes ◽  
Cultivation System ◽  
Mammalian Infection

ABSTRACT Lyme disease is a tick-borne infection that can lead to chronic, debilitating problems if not recognized or treated appropriately. Borrelia burgdorferi, the causative agent of Lyme disease, is maintained in nature by a complex enzootic cycle involving Ixodes ticks and mammalian hosts. Many previous studies support the notion that B. burgdorferi differentially expresses numerous genes and proteins to help it adapt to growth in the mammalian host. In this regard, several studies have utilized a dialysis membrane chamber (DMC) cultivation system to generate “mammalian host-adapted” spirochetes for the identification of genes selectively expressed during mammalian infection. Here, we have exploited the DMC cultivation system in conjunction with microarray technology to examine the global changes in gene expression that occur in the mammalian host. To identify genes regulated by only mammal-specific signals and not by temperature, borrelial microarrays were hybridized with cDNA generated either from organisms temperature shifted in vitro from 23°C to 37°C or from organisms cultivated by using the DMC model system. Statistical analyses of the combined data sets revealed that 125 genes were expressed at significantly different levels in the mammalian host, with almost equivalent numbers of genes being up- or down-regulated by B. burgdorferi within DMCs compared to those undergoing temperature shift. Interestingly, during DMC cultivation, the vast majority of genes identified on the plasmids were down-regulated (79%), while the differentially expressed chromosomal genes were almost entirely up-regulated (93%). Global analysis of the upstream promoter regions of differentially expressed genes revealed that several share a common motif that may be important in transcriptional regulation during mammalian infection. Among genes with known or putative functions, the cell envelope category, which includes outer membrane proteins, was found to contain the most differentially expressed genes. The combined findings have generated a subset of genes that can now be further characterized to help define their role or roles with regard to B. burgdorferi virulence and Lyme disease pathogenesis.

scholarly journals Profiling of Temperature-Induced Changes in Borrelia burgdorferi Gene Expression by Using Whole Genome Arrays

2003 ◽  
Vol 71 (4) ◽  
pp. 1689-1705 ◽  
Author(s):  
Caroline Ojaimi ◽  
Chad Brooks ◽  
Sherwood Casjens ◽  
Patricia Rosa ◽  
Abdallah Elias ◽  
...  
Keyword(s):  
Gene Expression ◽  
Borrelia Burgdorferi ◽  
The United States ◽  
Etiologic Agent ◽  
Open Reading Frames ◽  
Differentially Expressed ◽  
Mammalian Host ◽  
Regulated Expression ◽  
Two Temperatures ◽  
Induced Changes

ABSTRACT Borrelia burgdorferi is the etiologic agent of Lyme disease, the most prevalent arthropod-borne disease in the United States. The genome of the type strain, B31, consists of a 910,725-bp linear chromosome and 21 linear and circular plasmids comprising 610,694 bp. During its life cycle, the spirochete exists in distinctly different environments, cycling between a tick vector and a mammalian host. Temperature is one environmental factor known to affect B. burgdorferi gene expression. To identify temperature-responsive genes, genome arrays containing 1,662 putative B. burgdorferi open reading frames (ORFs) were prepared on nylon membranes and employed to assess gene expression in B. burgdorferi B31 grown at 23 and 35°C. Differences in expression of more than 3.5 orders of magnitude could be readily discerned and quantitated. At least minimal expression from 91% of the arrayed ORFs could be detected. A total of 215 ORFs were differentially expressed at the two temperatures; 133 were expressed at significantly greater levels at 35°C, and 82 were more significantly expressed at 23°C. Of these 215 ORFs, 134 are characterized as genes of unknown function. One hundred thirty-six (63%) of the differentially expressed genes are plasmid encoded. Of particular interest is plasmid lp54 which contains 76 annotated putative genes; 31 of these exhibit temperature-regulated expression. These findings underscore the important role plasmid-encoded genes may play in adjustment of B. burgdorferi to growth under diverse environmental conditions.

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