scholarly journals Mutations in the lspA1 and lspA2 Genes of Haemophilus ducreyi Affect the Virulence of This Pathogen in an Animal Model System

2003 ◽  
Vol 71 (5) ◽  
pp. 2478-2486 ◽  
Author(s):  
Christine K. Ward ◽  
Jo L. Latimer ◽  
Joseph Nika ◽  
Merja Vakevainen ◽  
Jason R. Mock ◽  
...  
Keyword(s):  
Animal Model ◽  
Parent Strain ◽  
Type Strain ◽  
Double Mutant ◽  
Wild Type Strain ◽  
Culture Supernatant ◽  
Haemophilus Ducreyi ◽  
Supernatant Fluid ◽  
Wild Type ◽  
Wild Type Parent

ABSTRACT Haemophilus ducreyi 35000HP contains two genes, lspA1 and lspA2, whose predicted protein products have molecular weights of 456,000 and 543,000, respectively (C. K. Ward, S. R. Lumbley, J. L. Latimer, L. D. Cope, and E. J. Hansen, J. Bacteriol. 180:6013-6022, 1998). We have constructed three H. ducreyi 35000HP mutants containing antibiotic resistance cartridges in one or both of the lspA1 and lspA2 open reading frames. Western blot analysis using LspA1- and LspA2-specific monoclonal antibodies indicated that the wild-type parent strain 35000HP expressed LspA1 protein that was readily detectable in culture supernatant fluid together with a barely detectable amount of LspA2 protein. The lspA2 mutant 35000HP.2 expressed LspA1 protein that was detectable in culture supernatant fluid and no LspA2 protein. In contrast, the H. ducreyi lspA1 mutant 35000HP.1, which did not express the LspA1 protein, expressed a greater quantity of the LspA2 protein than did the wild-type parent strain. The lspA1 lspA2 double mutant 35000HP.12 expressed neither LspA1 nor LspA2. The three mutant strains adhered to human foreskin fibroblasts and to a human keratinocyte cell line in vitro at a level that was not significantly different from that of the wild-type strain 35000HP. Lack of expression of the LspA1 protein by both the lspA1 mutant and the lspA1 lspA2 double mutant was associated with an increased tendency to autoagglutinate. When evaluated in the temperature-dependent rabbit model for chancroid, the lspA1 lspA2 double mutant was substantially less virulent than the wild-type strain 35000HP. The results of these studies indicated that H. ducreyi requires both the LspA1 and LspA2 proteins to be fully virulent in this animal model for experimental chancroid.

scholarly journals Mapping the Role of AcrAB-TolC Efflux Pumps in the Evolution of Antibiotic Resistance Reveals Near-MIC Treatments Facilitate Resistance Acquisition

2020 ◽  
Author(s):  
Ariel M. Langevin ◽  
Imane El Meouche ◽  
Mary J. Dunlop
Keyword(s):  
Antibiotic Resistance ◽  
Genetic Background ◽  
Parent Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Efflux Pumps ◽  
Wild Type ◽  
Antibiotic Concentration ◽  
Drug Concentrations ◽  
Evolution Of Resistance

ABSTRACTAntibiotic resistance has become a major public health concern as bacteria evolve to evade drugs, leading to recurring infections and a decrease in antibiotic efficacy. Systematic efforts have revealed mechanisms involved in resistance; yet, in many cases, how these specific mechanisms accelerate or slow the evolution of resistance remains unclear. Here, we conducted a systematic study of the impact of the AcrAB-TolC efflux pump on the evolution of antibiotic resistance. We mapped how population growth rate and resistance change over time as a function of both the antibiotic concentration and the parent strain’s genetic background. We compared the wild type strain to a strain overexpressing AcrAB-TolC pumps and a strain lacking functional pumps. In all cases, resistance emerged when cultures were treated with chloramphenicol concentrations near the MIC of their respective parent strain. The genetic background of the parent strain also influenced resistance acquisition. The wild type strain evolved resistance within 24 h through mutations in the acrAB operon and its associated regulators. Meanwhile, the strain overexpressing AcrAB-TolC evolved resistance more slowly than the wild type strain; this strain achieved resistance in part through point mutations in acrB and the acrAB promoter. Surprisingly, the strain without functional AcrAB-TolC efflux pumps still gained resistance, which it achieved through upregulation of redundant efflux pumps. Overall, our results suggest that treatment conditions just above the MIC pose the largest risk for the evolution of resistance and that AcrAB-TolC efflux pumps impact the pathway by which chloramphenicol resistance is achieved.IMPORTANCECombatting the rise of antibiotic resistance is a significant challenge. Efflux pumps are an important contributor to drug resistance; they exist across many cell types and can export numerous classes of antibiotics. Cells can regulate pump expression to maintain low intracellular drug concentrations. Here, we explored how resistance emerged depending on the antibiotic concentration, as well as the presence of efflux pumps and their regulators. We found that treatments near antibiotic concentrations that inhibit the parent strain’s growth were most likely to promote resistance. While wild type, pump overexpression, and pump knock out strains were all able to evolve resistance, they differed in the absolute level of resistance evolved, the speed at which they achieved resistance, and the genetic pathways involved. These results indicate that specific treatment regimens may be especially problematic for the evolution of resistance and that the strain background can influence how resistance is achieved.

scholarly journals Quorum Sensing Regulates Exopolysaccharide Production, Motility, and Virulence in Pseudomonas syringae

2005 ◽  
Vol 18 (7) ◽  
pp. 682-693 ◽  
Author(s):  
Beatriz Quiñones ◽  
Glenn Dulla ◽  
Steven E. Lindow
Keyword(s):  
Quorum Sensing ◽  
Pseudomonas Syringae ◽  
Type Strain ◽  
Double Mutant ◽  
Wild Type Strain ◽  
Homoserine Lactone ◽  
Brown Spot ◽  
Wild Type ◽  
In Planta ◽  
Alginate Production

The N-acyl homoserine lactone (AHL)-mediated quorumsensing system in the phytopathogen Pseudomonas syringae pv. syringae requires the AHL synthase AhlI and the regulator AhlR, and is additionally subject to regulation by AefR. The contribution of quorum sensing to the expression of a variety of traits expected to be involved in epiphytic fitness and virulence of P. syringae were examined. Both an aefR- mutant and an ahlR- double mutant, deficient in AHL production, were significantly impaired in alginate production and had an increased susceptibility to hydrogen peroxide compared with the wild-type strain. These mutants were hypermotile in culture, invaded leaves more rapidly, and caused an increased incidence of brown spot lesions on bean leaves after a 48-h moist incubation. Interestingly, an aefR- mutant was both the most motile and virulent. Like the wild-type strain, the AHL-deficient mutant strains incited water-soaked lesions on bean pods. However, lesions caused by an ahlI- ahlR- double mutant were larger, whereas those incited by an aefR- mutant were smaller. In contrast, tissue maceration of pods, which occurs at a later stage of infection, was completely abolished in the AHL-deficient mutants. Both the incidence of disease and in planta growth of P. syringae pv. tabaci were greatly reduced in transgenic tobacco plants that produced AHL compared with wild-type plants. These results demonstrate that quorum sensing in P. syringae regulates traits that contribute to epiphytic fitness as well as to distinct stages of disease development during plant infection.

scholarly journals Novel Toluene Elimination System in a Toluene-Tolerant Microorganism

2000 ◽  
Vol 182 (22) ◽  
pp. 6451-6455 ◽  
Author(s):  
Hideki Kobayashi ◽  
Katsuyuki Uematsu ◽  
Hisako Hirayama ◽  
Koki Horikoshi
Keyword(s):  
Membrane Proteins ◽  
Cell Membrane ◽  
Outer Membrane ◽  
Parent Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Outer Membrane Proteins ◽  
Membrane Vesicles ◽  
Wild Type ◽  
Sensitive Mutant

ABSTRACT In studies of Pseudomonas putida IH-2000, a toluene-tolerant microorganism, membrane vesicles (MVs) were found to be released from the outer membrane when toluene was added to the culture. These MVs were found to be composed of phospholipids, lipopolysaccharides (LPS), and very low amounts of outer membrane proteins. The MVs also contained a higher concentration of toluene molecules (0.172 ± 0.012 mol/mol of lipid) than that found in the cell membrane. In contrast to the wild-type strain, the toluene-sensitive mutant strain 32, which differs from the parent strain in LPS and outer membrane proteins, did not release MVs from the outer membrane. The toluene molecules adhering to the outer membrane are eliminated by the shedding of MVs, and this system appears to serve as an important part of the toluene tolerance system of IH-2000.

scholarly journals Inhibition of Phagocytosis by Haemophilus ducreyi Requires Expression of the LspA1 and LspA2 Proteins

2003 ◽  
Vol 71 (10) ◽  
pp. 5994-6003 ◽  
Author(s):  
Merja Vakevainen ◽  
Steven Greenberg ◽  
Eric J. Hansen
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Rabbit Model ◽  
Inhibitory Effect ◽  
Haemophilus Ducreyi ◽  
Bacterial Cells ◽  
Wild Type ◽  
Temperature Dependent ◽  
Live Bacterial Cells

ABSTRACT Haemophilus ducreyi previously has been shown to inhibit the phagocytosis of both secondary targets and itself by certain cells in vitro. Wild-type H. ducreyi strain 35000HP contains two genes, lspA1 and lspA2, whose encoded protein products are predicted to be 456 and 543 kDa, respectively. An isogenic mutant of H. ducreyi 35000HP with inactivated lspA1 and lspA2 genes has been shown to exhibit substantially decreased virulence in the temperature-dependent rabbit model for chancroid. This lspA1 lspA2 mutant was tested for its ability to inhibit phagocytosis of immunoglobulin G-opsonized particles by differentiated HL-60 and U-937 cells and by J774A.1 cells. The wild-type strain H. ducreyi 35000HP readily inhibited phagocytosis, whereas the lspA1 lspA2 mutant was unable to inhibit phagocytosis. Similarly, the wild-type strain was resistant to phagocytosis, whereas the lspA1 lspA2 mutant was readily engulfed by phagocytes. This inhibitory effect of wild-type H. ducreyi on phagocytic activity was primarily associated with live bacterial cells but could also be found, under certain conditions, in concentrated H. ducreyi culture supernatant fluids that lacked detectable outer membrane fragments. Both the wild-type strain and the lspA1 lspA2 mutant attached to phagocytes at similar levels. These results indicate that the LspA1 and LspA2 proteins of H. ducreyi are involved, directly or indirectly, in the antiphagocytic activity of this pathogen, and they provide a possible explanation for the greatly reduced virulence of the lspA1 lspA2 mutant.

scholarly journals Functional Role of Bradyrhizobium japonicum Trehalose Biosynthesis and Metabolism Genes during Physiological Stress and Nodulation

2009 ◽  
Vol 76 (4) ◽  
pp. 1071-1081 ◽  
Author(s):  
Masayuki Sugawara ◽  
Eddie J. Cytryn ◽  
Michael J. Sadowsky
Keyword(s):  
Bradyrhizobium Japonicum ◽  
Type Strain ◽  
Double Mutant ◽  
Wild Type Strain ◽  
Physiological Stress ◽  
Desiccation Stress ◽  
Wild Type ◽  
Trehalose Biosynthesis ◽  
Soybean Plants ◽  
Trehalose Accumulation

ABSTRACT Trehalose, a disaccharide accumulated by many microorganisms, acts as a protectant during periods of physiological stress, such as salinity and desiccation. Previous studies reported that the trehalose biosynthetic genes (otsA, treS, and treY) in Bradyrhizobium japonicum were induced by salinity and desiccation stresses. Functional mutational analyses indicated that disruption of otsA decreased trehalose accumulation in cells and that an otsA treY double mutant accumulated an extremely low level of trehalose. In contrast, trehalose accumulated to a greater extent in a treS mutant, and maltose levels decreased relative to that seen with the wild-type strain. Mutant strains lacking the OtsA pathway, including the single, double, and triple ΔotsA, ΔotsA ΔtreS and ΔotsA ΔtreY, and ΔotsA ΔtreS ΔtreY mutants, were inhibited for growth on 60 mM NaCl. While mutants lacking functional OtsAB and TreYZ pathways failed to grow on complex medium containing 60 mM NaCl, there was no difference in the viability of the double mutant strain when cells were grown under conditions of desiccation stress. In contrast, mutants lacking a functional TreS pathway were less tolerant of desiccation stress than the wild-type strain. Soybean plants inoculated with mutants lacking the OtsAB and TreYZ pathways produced fewer mature nodules and a greater number of immature nodules relative to those produced by the wild-type strain. Taken together, results of these studies indicate that stress-induced trehalose biosynthesis in B. japonicum is due mainly to the OtsAB pathway and that the TreS pathway is likely involved in the degradation of trehalose to maltose. Trehalose accumulation in B. japonicum enhances survival under conditions of salinity stress and plays a role in the development of symbiotic nitrogen-fixing root nodules on soybean plants.

scholarly journals Diffusible Signal Factor–Repressed Extracellular Traits Enable Attachment of Xylella fastidiosa to Insect Vectors and Transmission

2014 ◽  
Vol 104 (1) ◽  
pp. 27-33 ◽  
Author(s):  
Clelia Baccari ◽  
Nabil Killiny ◽  
Michael Ionescu ◽  
Rodrigo P. P. Almeida ◽  
Steven E. Lindow
Keyword(s):  
Type Strain ◽  
Host Plants ◽  
Wild Type Strain ◽  
Culture Supernatant ◽  
Culture Media ◽  
Xylella Fastidiosa ◽  
Xylem Sap ◽  
Wild Type ◽  
Diffusible Signal Factor ◽  
Type Strains

The hypothesis that a wild-type strain of Xylella fastidiosa would restore the ability of rpfF mutants blocked in diffusible signal factor production to be transmitted to new grape plants by the sharpshooter vector Graphocephala atropunctata was tested. While the rpfF mutant was very poorly transmitted by vectors irrespective of whether they had also fed on plants infected with the wild-type strain, wild-type strains were not efficiently transmitted if vectors had fed on plants infected with the rpfF mutant. About 100-fewer cells of a wild-type strain attached to wings of a vector when suspended in xylem sap from plants infected with an rpfF mutant than in sap from uninfected grapes. The frequency of transmission of cells suspended in sap from plants that were infected by the rpfF mutant was also reduced over threefold. Wild-type cells suspended in a culture supernatant of an rpfF mutant also exhibited 10-fold less adherence to wings than when suspended in uninoculated culture media. A factor released into the xylem by rpfF mutants, and to a lesser extent by the wild-type strain, thus inhibits their attachment to, and thus transmission by, sharpshooter vectors and may also enable them to move more readily through host plants.

scholarly journals Characterization of a Haemophilus ducreyi Mutant Deficient in Expression of Cytolethal Distending Toxin

1999 ◽  
Vol 67 (8) ◽  
pp. 3900-3908 ◽  
Author(s):  
Marla K. Stevens ◽  
Jo L. Latimer ◽  
Sheryl R. Lumbley ◽  
Christine K. Ward ◽  
Leslie D. Cope ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Hela Cells ◽  
Culture Supernatant ◽  
Haemophilus Ducreyi ◽  
Cytolethal Distending Toxin ◽  
Supernatant Fluid ◽  
Hacat Keratinocytes ◽  
Wild Type ◽  
Insertional Inactivation

ABSTRACT Haemophilus ducreyi expresses a soluble cytolethal distending toxin (CDT) that kills HeLa, HEp-2, and other human epithelial cells in vitro. H. ducreyi CDT activity is encoded by a three-gene cluster (cdtABC), and antibody to the cdtC gene product can neutralize CDT activity in vitro (L. D. Cope, S. R. Lumbley, J. L. Latimer, J. Klesney-Tait, M. K. Stevens, L. S. Johnson, M. Purven, R. S. Munson, Jr., T. Lagergard, J. D. Radolf, and E. J. Hansen, Proc. Natl. Acad. Sci. USA 94:4056–4061, 1997). Culture supernatant fluid from a recombinant Escherichia colistrain containing the H. ducreyi cdtABC gene cluster readily killed both HeLa cells and HaCaT keratinocytes and had a modest inhibitory effect on the growth of human foreskin fibroblasts. Insertional inactivation of the cdtC gene in this recombinant E. coli strain eliminated the ability of this strain to kill HeLa cells and HaCaT keratinocytes. This mutatedH. ducreyi cdtABC gene cluster was used to construct an isogenic H. ducreyi cdtC mutant. Monoclonal antibodies against the H. ducreyi CdtA, CdtB, and CdtC proteins were used to characterize protein expression by this cdtCmutant. Culture supernatant fluid from this H. ducreyi cdtCmutant did not detectably affect any of the human cells used in this study. The presence of the wild-type H. ducreyi cdtC gene in trans in this H. ducreyi mutant restored its ability to express a CDT that killed both HeLa cells and HaCaT keratinocytes. The isogenic H. ducreyi cdtC mutant was shown to be as virulent as its wild-type parent strain in the temperature-dependent rabbit model for experimental chancroid. Lack of expression of the H. ducreyi CdtC protein also did not affect the ability of this H. ducreyi mutant to survive in the skin of rabbits.

scholarly journals The galE Gene of Campylobacter jejuni Is Involved in Lipopolysaccharide Synthesis and Virulence

2000 ◽  
Vol 68 (5) ◽  
pp. 2594-2601 ◽  
Author(s):  
Benjamin N. Fry ◽  
Shi Feng ◽  
Yuen-Yuen Chen ◽  
Diane G. Newell ◽  
Peter J. Coloe ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Parent Strain ◽  
Type Strain ◽  
Essential Role ◽  
Lipid A ◽  
Wild Type Strain ◽  
Gram Negative Bacteria ◽  
Wild Type ◽  
Gram Negative ◽  
Important Virulence Factor

ABSTRACT Lipopolysaccharide (LPS) is one of the main virulence factors of gram-negative bacteria. The LPS from Campylobacter spp. has endotoxic properties and has been shown to play a role in adhesion. We previously cloned a gene cluster (wla) which is involved in the synthesis of the Campylobacter jejuni 81116 LPS molecule. Sequence alignment of the first gene in this cluster indicated similarity with galE genes. These genes encode a UDP-glucose 4-epimerase, which catalyzes the interconversion of UDP-galactose and UDP-glucose. A Salmonella galE mutant was transformed with the galE gene from C. jejuni. The LPS analysis of wild-type, galE, and complementedgalE Salmonella strains showed that the C. jejuni galE gene could restore the smooth wild-typeSalmonella LPS. A UDP-glucose 4-epimerase assay was used to demonstrate that the galE gene from C. jejuniencoded this epimerase. We constructed a C. jejuni galEmutant which expressed a lipid A-core molecule of reduced molecular weight that did not react with antiserum raised against the parental strain. These results show an essential role for the galEgene in the synthesis of C. jejuni LPS. ThegalE mutant also showed a reduction in its ability to adhere to and invade INT407 cells. However, it was still able to colonize chickens to the same level as the wild-type strain. The serum resistance and hemolytic activity of this mutant were not changed compared to the parent strain. The ability of the mutant to take up DNA and integrate it in its genome was reduced 20-fold. These results show that LPS of C. jejuni is an important virulence factor.

scholarly journals Expression of the LspA1 and LspA2 Proteins by Haemophilus ducreyi Is Required for Virulence in Human Volunteers

2004 ◽  
Vol 72 (8) ◽  
pp. 4528-4533 ◽  
Author(s):  
Diane M. Janowicz ◽  
Kate R. Fortney ◽  
Barry P. Katz ◽  
Jo L. Latimer ◽  
Kaiping Deng ◽  
...  
Keyword(s):  
Polymorphonuclear Leukocytes ◽  
Double Mutant ◽  
Human Subjects ◽  
Rabbit Model ◽  
Haemophilus Ducreyi ◽  
Wild Type ◽  
Human Volunteers ◽  
Mean Size ◽  
Wild Type Parent

ABSTRACT Haemophilus ducreyi colocalizes with polymorphonuclear leukocytes and macrophages and evades phagocytosis during experimental infection of human volunteers. H. ducreyi contains two genes, lspA1 and lspA2, which encode predicted proteins of 456 and 543 kDa, respectively. Compared to its wild-type parent, an lspA1 lspA2 double mutant does not inhibit phagocytosis by macrophage and myelocytic cell lines in vitro and is attenuated in an experimental rabbit model of chancroid. To test whether expression of LspA1 and LspA2 was necessary for virulence in humans, six volunteers were experimentally infected. Each volunteer was inoculated with three doses (ranging from 85 to 112 CFU) of the parent (35000HP) in one arm and three doses (ranging from 60 to 822 CFU) of the mutant (35000HPΩ12) in the other arm. The papule formation rates were 88% (95% confidence interval [95% CI], 76.8 to 99.9%) at 18 parent sites and 72% (95% CI, 44.4 to 99.9%) at 18 mutant sites (P = 0.19). However, papules were significantly smaller at mutant sites (mean size, 24.8 mm2) than at parent sites (mean size, 39.1 mm2) 24 h after inoculation (P = 0.0002). The pustule formation rates were 44% (95% CI, 5.8 to 77.6%) at parent sites and 0% (95% CI, 0 to 39.4%) at mutant sites (P = 0.009). With the caveat that biosafety regulations preclude testing of a complemented mutant in human subjects, these results indicate that expression of LspA1 and LspA2 facilitates the ability of H. ducreyi to initiate disease and to progress to pustule formation in humans.

scholarly journals Role of Phosphoglucomutase of Stenotrophomonasmaltophilia in Lipopolysaccharide Biosynthesis, Virulence, and Antibiotic Resistance

2003 ◽  
Vol 71 (6) ◽  
pp. 3068-3075 ◽  
Author(s):  
Geoffrey A. McKay ◽  
Donald E. Woods ◽  
Kelly L. MacDonald ◽  
Keith Poole
Keyword(s):  
Animal Model ◽  
Antibiotic Resistance ◽  
Parent Strain ◽  
Antimicrobial Agents ◽  
Bifunctional Enzyme ◽  
Wild Type ◽  
Lipopolysaccharide Biosynthesis ◽  
Wild Type Parent ◽  
Alginate Biosynthesis

ABSTRACT A homologue of the algC gene, responsible for the production of a phosphoglucomutase (PGM) associated with LPS and alginate biosynthesis in Pseudomonas aeruginosa, spgM, was cloned from Stenotrophomonas maltophilia. The spgM gene was shown to encode a bifunctional enzyme with both PGM and phosphomannomutase activities. Mutants lacking spgM produced less LPS than the SpgM+ parent strain and had a tendency for shorter O polysaccharide chains. No changes in LPS chemistry were obvious as a result of the loss of spgM. Significantly, however, spgM mutants displayed a modest increase in susceptibility to several antimicrobial agents and were completely avirulent in an animal model of infection. The latter finding may relate to the resultant serum sensitivity of spgM mutants which, unlike the wild-type parent strain, were rapidly killed by human serum. These data highlight the contribution made by LPS to the antimicrobial resistance and virulence of S. maltophilia.

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