[Show abstract][Hide abstract] ABSTRACT: RpoS, one of the two alternative σ factors in Borrelia burgdorferi, is tightly controlled by multiple regulators and, in turn, determines expression of many critical virulence factors. Here we show that increasing RpoS expression causes cell death. The immediate effect of increasing RpoS expression was to promote bacterial division and as a consequence result in a rapid increase in cell number before causing bacterial death. No DNA fragmentation or degradation was observed during this induced cell death. Cryo-electron microscopy showed induced cells first formed blebs, which were eventually released from dying cells. Apparently blebbing initiated cell disintegration leading to cell death. These findings led us to hypothesize that increasing RpoS expression triggers intracellular programs and/or pathways that cause spirochete death. The potential biological significance of induced cell death may help B. burgdorferi regulate its population to maintain its life cycle in nature.
PLoS ONE 01/2013; 8(12):e83276. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Since the RpoN-RpoS regulatory network was revealed in the Lyme disease spirochete Borrelia burgdorferi a decade ago, both upstream and downstream of the pathway have been intensively investigated. While significant progress has been made into understanding of how the network is regulated, most notably, discovering a relationship of the network with Rrp2 and BosR, only three crucial virulence factors, including outer surface protein C (OspC) and decorin-binding proteins (Dbps) A and B, are associated with the pathway. Moreover, for more than 10 years no single RpoS-controlled gene has been found to be critical for infection, raising a question about whether additional RpoS-dependent virulence factors remain to be identified.
The rpoS gene was deleted in B. burgdorferi; resulting mutants were modified to constitutively express all the known virulence factors, OspC, DbpA and DbpB. This genetic modification was unable to restore the rpoS mutant with infectivity.
The inability to restore the rpoS mutant with infectivity by simultaneously over-expressing all the three virulence factors allows us to conclude RpoS also regulates essential genes that remain to be identified in B. burgdorferi.
PLoS ONE 01/2012; 7(12):e53212. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The gene bb0250 of Borrelia burgdorferi is a homolog of the dedA family, encoding integral inner membrane proteins that are present in nearly all species of bacteria. To date, no precise function has been attributed to any dedA gene. Unlike many bacterial species, such as Escherichia coli, which has eight dedA genes, B. burgdorferi possesses only one, annotated bb0250, providing a unique opportunity to investigate the functions of the dedA family. Here, we show that bb0250 is able to restore normal growth and cell division to a temperature-sensitive E. coli mutant with simultaneous deletions of two dedA genes, yqjA and yghB, and encodes a protein that localizes to the inner membrane of E. coli. The bb0250 gene could be deleted from B. burgdorferi only after introduction of a promoterless bb0250 under the control of an inducible lac promoter, indicating that it is an essential gene in this organism. Growth of the mutant in the absence of isopropyl-β-d-thiogalactopyranoside resulted in cell death, preceded by cell division defects characterized by elongated cells and membrane bulges, demonstrating that bb0250 is required for proper cell division and envelope integrity. Finally, we show that BB0250 depletion leads to imbalanced membrane phospholipid composition in borrelia. These results demonstrate a strong conservation of function of the dedA gene family across diverse species of Gram-negative bacteria and a requirement for this protein family for normal membrane lipid composition and cell division.
Journal of bacteriology 12/2010; 192(23):6105-15. · 3.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: During cycling between the tick vector and a mammal, the Lyme disease spirochaete Borrelia burgdorferi must coordinate expression of outer-surface proteins (Osps) A and B to quickly respond to environmental changes. The pathogen abundantly produces OspA/B in the tick, but represses their expression during mammalian infection. This paper reports a regulatory structure, consisting of two sequences flanking the ospAB promoter, that is required for enhancing ospA expression in B. burgdorferi grown in vitro, but repressing its expression during murine infection. Deletion or replacement of either the upstream or downstream sequence of the ospAB promoter caused a significant decrease in ospA expression in vitro, but a dramatic increase during murine infection. Fusion of either sequence with the flaB reporter promoter led to increased expression of an ospA reporter gene in vitro, but a decrease in the murine host. Furthermore, simultaneous fusion of both sequences with the reporter promoter showed a synergistic effect in enhancing expression of the ospA reporter in vitro, but repressing its expression during murine infection. Taken together, the results demonstrate that the regulatory structure functions oppositely in the two different environments and potentially provides B. burgdorferi with a molecular mechanism to quickly adapt to the distinct environments during its enzootic life cycle.
[Show abstract][Hide abstract] ABSTRACT: The Lyme disease spirochete Borrelia burgdorferi dramatically upregulates outer surface protein C (OspC) in response to fresh bloodmeal during transmission from the tick vector to a mammal, and abundantly produces the antigen during early infection. As OspC is an effective immune target, to evade the immune system B. burgdorferi downregulates the antigen once the anti-OspC humoral response has developed, suggesting an important role for OspC during early infection.
In this study, a borrelial mutant producing an OspC antigen with a 5-amino-acid deletion was generated. The deletion didn't significantly increase the 50% infectious dose or reduce the tissue bacterial burden during infection of the murine host, indicating that the truncated OspC can effectively protect B. burgdorferi against innate elimination. However, the deletion greatly impaired the ability of B. burgdorferi to disseminate to remote tissues after inoculation into mice.
The study indicates that OspC plays an important role in dissemination of B. burgdorferi during mammalian infection.
PLoS ONE 01/2010; 5(12):e15830. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The surface lipoproteins of the Lyme disease spirochaete Borrelia burgdorferi directly interact with tissue microenvironments during mammalian infection, and thus potentially affect various aspects of infection. To investigate the influence of surface antigen synthesis on infectious behaviour, B. burgdorferi was modified to constitutively produce the well-characterized surface lipoproteins OspA and invariant VlsE. Although increasing OspA or VlsE production did not significantly affect synthesis of other surface lipoproteins or spirochaetal growth in vitro, overexpressing vlsE resulted in increased ospA but decreased ospC expression, and overexpressing ospA led to decreased ospC and vlsE expression in severe combined immunodeficient (SCID) mice. Increasing the expression of either ospA or vlsE did not alter the ID(50), but affected spirochaetal dissemination and significantly reduced tissue spirochaete loads in SCID mice. In immunocompetent mice, increased vlsE expression resulted in quick clearance of infection, while constitutive ospA expression led to a substantial ID(50) increase and severely impaired dissemination. Furthermore, B. burgdorferi with constitutive ospA expression persisted in the skin tissue but was cleared from both heart and joints of chronically infected immunocompetent mice. Taken together, the study indicates that increasing production of OspA or invariant VlsE influences lipoprotein gene expression in the murine host and alters the infectious behaviour of B. burgdorferi.
[Show abstract][Hide abstract] ABSTRACT: The Lyme disease spirochete Borrelia burgdorferi must repress expression of outer surface protein C (OspC) to effectively evade specific humoral immunity and to establish persistent infection. This ability largely relies upon a regulatory element, the only operator that has been reported in spirochetal bacteria. Immediately upstream of the ospC promoter, two sets of inverted repeats (IRs) constitute small and large palindromes, in which the right IR of the large palindrome contains the left IR of the small one, and may collectively function as the ospC operator. In the study, the large palindrome with or without the small IR was fused with an flaB promoter, which was used to drive expression of a promoterless ospC copy as a reporter gene, and introduced into OspC-deficient B. burgdorferi. The presence of the large palindrome alone significantly reduced ospC expression driven by the fused flaB promoter in the joint tissue of severe combined immunodeficiency (SCID) mice, and rescued spirochetes from elimination by passively transferred OspC antibody in infected SCID mice and specific immune responses elicited in immunocompetent mice, confirming a function of the IRs as an operator. Inclusion of the small IR further enhanced the ability of the large palindrome to reduce the activity of the fused flaB promoter, indicating that the small IR is a part of the operator. Taken together, the study led to successful verification and dissection of the ospC operator.
[Show abstract][Hide abstract] ABSTRACT: To initiate infection, a microbial pathogen must be able to evade innate immunity. Here we show that the Lyme disease spirochete Borrelia burgdorferi depends on its surface lipoproteins for protection against innate defences. The deficiency for OspC, an abundantly expressed surface lipoprotein during early infection, led to quick clearance of B. burgdorferi after inoculation into the skin of SCID mice. Increasing expression of any of the four randomly chosen surface lipoproteins, OspA, OspE, VlsE or DbpA, fully protected the ospC mutant from elimination from the skin tissue of SCID mice; moreover, increased OspA, OspE or VlsE expression allowed the mutant to cause disseminated infection and restored the ability to effectively colonize both joint and skin tissues, albeit the dissemination process was much slower than that of the mutant restored with OspC expression. When the ospC mutant was modified to express OspA under control of the ospC regulatory elements, it registered only a slight increase in the 50% infectious dose than the control in SCID mice but a dramatic increase in immunocompetent mice. Taken together, the study demonstrated that the surface lipoproteins provide B. burgdorferi with an essential protective function against host innate elimination.
[Show abstract][Hide abstract] ABSTRACT: Both decorin-binding proteins (DbpA and DbpB) of the Lyme disease spirochete Borrelia burgdorferi bind decorin and glycosaminoglycans, two important building blocks of proteoglycans that are abundantly found in the extracellular matrix (ECM) and connective tissues as well as on cell surfaces of mammals. As an extracellular pathogen, B. burgdorferi resides primarily in the ECM and connective tissues and between host cells during mammalian infection. The interactions of B. burgdorferi with these host ligands mediated by DbpA and DbpB potentially influence various aspects of infection. Here, we show that both DbpA and DbpB are critical for the overall virulence of B. burgdorferi in the murine host. Disruption of the dbpBA locus led to nearly a 10(4)-fold increase in the 50% infectious dose (ID50). Complementation of the mutant with either dbpA or dbpB reduced the ID50 from over 10(4) to roughly 10(3) organisms. Deletion of the dbpBA locus affected colonization in all tissues of infected mice. The lack of dbpA alone precluded the pathogen from colonizing the heart tissue, and B. burgdorferi deficient for DbpB was recovered only from 42% of the heart specimens of infected mice. Although B. burgdorferi lacking either dbpA or dbpB was consistently grown from joint specimens of almost all infected mice, it generated bacterial loads significantly lower than the control. The deficiency in either DbpA or DbpB did not reduce the bacterial load in skin, but lack of both significantly did. Taken together, the study results indicate that neither DbpA nor DbpB is essential for mammalian infection but that both are critical for the overall virulence of B. burgdorferi.
Infection and immunity 04/2008; 76(3):1239-46. · 4.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: As an extracellular bacterium, the Lyme disease spirochete Borrelia burgdorferi resides primarily in the extracellular matrix and connective tissues and between host cells during mammalian infection, where decorin and glycosaminoglycans are abundantly found, so its interactions with these host ligands potentially affect various aspects of infection. Decorin-binding proteins (Dbps) A and B, encoded by a 2-gene operon, are outer surface lipoproteins with similar molecular weights and share approximately 40% identity, and both bind decorin and glycosaminoglycans. To investigate how DbpA and DbpB contribute differently to the overall virulence of B. burgdorferi, a dbpAB mutant was modified to overproduce the adhesins. Overproduction of either DbpA or DbpB resulted in restoration of the infectivity of the mutant to the control level, measured by 50% infectious dose (ID(50)), indicating that the two virulence factors are interchangeable in this regard. Overproduction of DbpA also allowed the mutant to disseminate to some but not all distal tissues slightly slower than the control, but the mutant with DbpB overproduction showed severely impaired dissemination to all tissues that were analyzed. The mutant with DbpA overproduction colonized all tissues, albeit generating bacterial loads significantly lower than the control in heart and joint, while the mutant overproducing DbpB remained severely defective in heart colonization and registered bacterial loads substantially lower than the control in joint. Taken together, the study indicated that DbpA and DbpB play a similar role in contribution to infectivity as measured by ID(50) value but contribute differently to dissemination and tissue colonization.
PLoS ONE 02/2008; 3(10):e3340. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tight regulation of surface antigenic expression is crucial for the pathogenic strategy of the Lyme disease spirochete, Borrelia burgdorferi. Here, we report the influence of increasing expression of decorin-binding protein A (DbpA), one of the most investigated spirochetal surface adhesins, on the 50% infectious dose (ID(50)), dissemination, tissue colonization, pathogenicity, and persistence of B. burgdorferi in the murine host. Our in vitro assays showed that increasing DbpA expression dramatically increased the interaction of B. burgdorferi with decorin and sensitivity to growth inhibition/killing by anti-DbpA antibodies; however, this increased interaction did not affect spirochetal growth and replication in the presence of decorin. Increasing DbpA expression significantly reduced ID(50) values and severely impaired dissemination in severe combined immunodeficiency (SCID) and immunocompetent mice. During infection of SCID mice, B. burgdorferi with increased DbpA expression was able to effectively colonize heart and skin tissues, but not joint tissues, completely abrogating arthritis virulence. Although increasing DbpA expression did not affect spirochetal persistence in the skin, it diminished the ability of B. burgdorferi to persist in the heart and joint tissues during chronic infection of immunocompetent mice. Taken together, the study highlights the importance of controlling surface antigen expression in the infectivity, dissemination, tissue colonization, pathogenicity, and persistence of B. burgdorferi during mammalian infection.
Infection and Immunity 10/2007; 75(9):4272-81. · 4.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Timely expression of the outer surface protein C (OspC) is crucial for the pathogenic strategy of the Lyme disease spirochete Borrelia burgdorferi. The pathogen abundantly expresses OspC during initial infection when the antigen is required, but downregulates when its presence poses a threat to the spirochetes once the anti-OspC humoral response has developed. Here, we show that a large palindromic sequence immediately upstream of the ospC promoter is essential for the repression of ospC expression during murine infection and for the ability of B. burgdorferi to evade specific OspC humoral immunity. Deletion of the sequence completely diminished the ability of B. burgdorferi to avoid clearance by transferred OspC antibody in SCID mice. B. burgdorferi lacking the regulatory element was able to initiate infection but unable to persist in immunocompetent mice. Taken together, the regulatory element immediately upstream of the ospC promoter serves as an operator that may interact with an unidentified repressor(s) to negatively regulate ospC expression and is essential for the immune evasion of B. burgdorferi.
[Show abstract][Hide abstract] ABSTRACT: Borrelia burgdorferi infection causes an initial skin lesion called erythema migrans (EM) in human Lyme disease and in models of monkey and rabbit borreliosis. EM results from the inflammatory response triggered by spirochete replication and likely develops to contain the initial infection but allows bacterial dissemination to occur. The essential lack of neutrophil involvement in EM histopathology prompted us to examine the consequence of increasing their recruitment in the inflammatory response to the Lyme disease agent. B. burgdorferi was modified genetically to constitutively express and secrete the chemokine KC, a neutrophil chemoattractant. After inoculation into the dermis of the murine host, control spirochetes induced an infiltration of macrophages, neutrophils, and basophils within 6 h; however, the recruited neutrophils and basophils were quickly substituted by eosinophils, and the inflammatory response became macrophage dominant by 16 h. Such a response failed to contain the initial infection and allowed the spirochetes to disseminate. In contrast, B. burgdorferi with KC secretion induced an intensive neutrophil infiltration at the inoculation site, and as a result, the host's ability to control the initial infection was greatly enhanced. Taken together, this study suggests that the failure of sufficient neutrophil recruitment and activation during the initial inflammatory response may allow B. burgdorferi to effectively colonize the mammalian host.
The Journal of Immunology 04/2007; 178(8):5109-15. · 5.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Lyme disease spirochete Borrelia burgdorferi expresses a broad array of adhesive molecules, including the decorin-binding proteins A and B (DbpA and DbpB), which are believed to play important roles in mammalian infection. The dbpBA locus was deleted; resulting mutants were able to infect both immunodeficient and immunocompetent mice, indicating that neither DbpA nor DbpB is essential for the infection of mammals, although the DbpAB deficiency may significantly attenuate infectivity potential.
Infection and Immunity 12/2006; 74(11):6509-12. · 4.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Lyme disease spirochete Borrelia burgdorferi reduces the expression of outer surface protein C (OspC) in response to the development of an anti-OspC humoral response, leading to the hypothesis that the ability to repress OspC expression is critical for the pathogen to proceed to chronic infection. B. burgdorferi was genetically modified to constitutively express OspC by introducing an extra ospC copy fused with the borrelial flagellar gene (flaB) promoter. Such a genetic modification did not reduce infectivity or pathogenicity in severe combined immunodeficiency mice but resulted in clearance of infection by passively transferred OspC antibody. Spirochetes with constitutive ospC expression were unable to establish chronic infections in immunocompetent mice unless they had undergone very destructive mutations in the introduced ospC copy. Two escape mutants were identified; one had all 7 bp deleted between the putative ribosome-binding site and the start codon, ATG, causing a failure in translational initiation, and the other mutant had an insertion of 2 bp between nucleotides 315 and 316, resulting in a nonsense mutation at codon 108. Thus, the ability of B. burgdorferi to repress ospC expression during mammalian infection allows the pathogen to avoid clearance and to preserve the integrity of the important gene for subsequent utilization during its enzootic life cycle.
Infection and Immunity 10/2006; 74(9):5177-84. · 4.07 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Borrelia burgdorferi, the Lyme disease spirochete, has a genome comprised of a linear chromosome and up to 21 plasmids. Loss of plasmids is associated with decreased infectivity and pathogenicity. Sixteen transformants were generated by transforming the noninfectious clone 5A13 with the recombinant plasmid pBBE22. The transformants were classified into nine groups based on plasmid content analysis. An infectivity study revealed that all nine transformants examined, each of which represented one of the plasmid patterns, were infectious in mice with severe combined immunodeficiency (SCID) regardless of their genomic compositions. Tissue bacterial quantification revealed that the loss of plasmids significantly reduced the spirochete burden in the heart and joint tissues, not in the skin, suggesting virulence factors may be tissue specific. Four transformants containing lp28-1 induced severe arthritis in SCID mice, in contrast to the five transformants lacking lp28-1. These pathogenicity studies associated lp28-1 with an arthritic phenotype and further studies may identify factors that contribute to arthritic pathology.
Infection and Immunity 12/2005; 73(11):7208-15. · 4.07 Impact Factor