Comparative proteome analysis of subcellular fractions from <b><i>Borrelia burgdorferi</i></b> by NEPHGE and IPG

Nowalk, Andrew; Nolder, Christi; Clifton, Dawn R.; Carroll, James A.

doi:10.1002/pmic.200500187

Cited by 56 publications

(73 citation statements)

References 46 publications

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“…The raw protein/peptide data from the LC-MS/MS analyses are provided in Table S1 in the supplemental material. The previously employed subcellular fractionation protocol (27)(28)(29)(30) was validated by the identification of well-known cellular markers. In the soluble fraction, we mapped and identified 50 proteins (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…These reannotated genomes were used to create a spreadsheet of all the proteins, with their molecular weights and isoelectric focusing points. The genome of Borrelia burgdorferi strain B31 was previously obtained from the J. Craig Venter Institute (JCVI) (29,33,34). These data were then used in GraphPad version 6 to generate graphs of each theoretical proteome (GraphPad Software, Inc., La Jolla, CA).…”

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confidence: 99%

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Multiple Posttranslational Modifications of Leptospira biflexa Proteins as Revealed by Proteomic Analysis

Stewart

Carroll

Olano

et al. 2016

Appl Environ Microbiol

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cThe saprophyte Leptospira biflexa is an excellent model for studying the physiology of the medically important Leptospira genus, the pathogenic members of which are more recalcitrant to genetic manipulation and have significantly slower in vitro growth. However, relatively little is known regarding the proteome of L. biflexa, limiting its utility as a model for some studies. Therefore, we have generated a proteomic map of both soluble and membrane-associated proteins of L. biflexa during exponential growth and in stationary phase. Using these data, we identified abundantly produced proteins in each cellular fraction and quantified the transcript levels from a subset of these genes using quantitative reverse transcription-PCR (RT-PCR). These proteins should prove useful as cellular markers and as controls for gene expression studies. We also observed a significant number of L. biflexa membrane-associated proteins with multiple isoforms, each having unique isoelectric focusing points. L. biflexa cell lysates were examined for several posttranslational modifications suggested by the protein patterns. Methylation and acetylation of lysine residues were predominately observed in the proteins of the membrane-associated fraction, while phosphorylation was detected mainly among soluble proteins. These three posttranslational modification systems appear to be conserved between the free-living species L. biflexa and the pathogenic species Leptospira interrogans, suggesting an important physiological advantage despite the varied life cycles of the different species. Members of the genus Leptospira are spirochetal bacteria that share a conserved spiral morphology and motility. Leptospires can be broadly divided into host-associated species (including pathogens and the closely related intermediate species [1]) and free-living saprophytes, and Picardeau et al. proposed that the host-associated species may have evolved from a free-living progenitor (2). Therefore, comparisons between species with different ecological niches may elucidate the mechanisms that allow host-associated species to infect, persist in, and sometimes cause fatal disease in their hosts.Leptospira biflexa, a free-living saprophyte, was discovered over 100 years ago (3) and is often used as a model organism for studies of the genus as a whole (2, 4-6). Although host-associated species, such as Leptospira interrogans, are more intensively studied, they exhibit a low transformation efficiency that approaches the limits of detection. The higher in vitro growth rate and more efficient genetic tools available to manipulate L. biflexa make this organism better suited for laboratory studies. Techniques such as targeted allelic exchange, transposon mutagenesis, conjugative transfer, the use of fluorescent markers, and the use of an inducible promoter system were first developed in L. biflexa before use in L. interrogans (5, 7-9). However, despite its being a model organism, there are significant gaps in our knowledge of L. biflexa. No large-scale microarray data o...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Multiple Posttranslational Modifications of Leptospira biflexa Proteins as Revealed by Proteomic Analysis

Stewart

Carroll

Olano

et al. 2016

Appl Environ Microbiol

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“…As can be seen, these organisms include Grampositive as well as Gram-negative (e.g., Borrelia burgdorferi [134]) bacteria. Most attention has focused on the cell surface enolase of group A streptococci.…”

Section: Moonlighting Bacterial Glycolytic Enzymesmentioning

confidence: 99%

Bacterial Virulence in the Moonlight: Multitasking Bacterial Moonlighting Proteins Are Virulence Determinants in Infectious Disease

2011

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2Men may not be able to multitask, but it is emerging that proteins can. This capacity of proteins to exhibit more than one function is termed protein moonlighting, and, surprisingly, many highly conserved proteins involved in metabolic regulation or the cell stress response have a range of additional biological actions which are involved in bacterial virulence. This review highlights the multiple roles exhibited by a range of bacterial proteins, such as glycolytic and other metabolic enzymes and molecular chaperones, and the role that such moonlighting activity plays in the virulence characteristics of a number of important human pathogens, including Staphylococcus aureus, Streptococcus pyogenes, Streptococcus pneumoniae, Helicobacter pylori, and Mycobacterium tuberculosis.

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“…To test the contribution of the BBA64 gene to the infectivity of B. burgdorferi for the mammalian host, it was necessary to confirm if different environmental signals induced comparable levels of expression of pgf 54 members in the strains to be used in the infectivity analysis (12,22,43). Immunoblot analysis using monospecific rat anti-BBA64 or anti-BBA66 (5) or monospecific mouse anti-BBA65 antibodies was used to detect the levels of these proteins.…”

Section: Construction Of the Bba64 Mtmentioning

confidence: 99%

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

et al. 2008

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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.Lyme disease is the most prevalent arthropod-borne infection in the United States and remains a significant public health issue in certain geographic loci (3,46). It is a multiphasic disorder with clinical symptoms involving the cutaneous, musculoskeletal, cardiovascular, and nervous systems (62). Borrelia burgdorferi, the causative agent of Lyme disease, is transmitted to several vertebrate hosts, including humans, by the bite of infected Ixodes ticks. When ticks consume a blood meal from mammalian hosts, there is a rapid alteration of gene expression in B. burgdorferi, facilitating adaptation of the spirochetes to the highly disparate environmental conditions that exist between the tick vector and the vertebrate host (4, 9, 10, 15, 29, 30-32, 44, 53, 57-59, 63, 67). This adaptive gene expression may aid in the efficient trafficking of the spirochetes from the tick vector to the mammalian host and subsequently facilitate dissemination and colonization of various host tissues (16,22).Whole-genome transcriptional analyses using B. burgdorferi, propagated under in vitro growth conditions that mimic either the tick vector or mammalian host environment, have revealed preferential expression of plasmid-encoded genes (4,44,53,66). Among the several linear and circular plasmids present in B. burgdorferi, linear plasmid 54 (lp54) encodes the largest number of open reading frames (ORFs) that exhibit differential gene expression in respon...

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Comparative proteome analysis of subcellular fractions from Borrelia burgdorferi by NEPHGE and IPG

Cited by 56 publications

References 46 publications

Multiple Posttranslational Modifications of Leptospira biflexa Proteins as Revealed by Proteomic Analysis

Multiple Posttranslational Modifications of Leptospira biflexa Proteins as Revealed by Proteomic Analysis

Bacterial Virulence in the Moonlight: Multitasking Bacterial Moonlighting Proteins Are Virulence Determinants in Infectious Disease

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

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