A (p)ppGpp-Null Mutant of Haemophilus ducreyi Is Partially Attenuated in Humans Due to Multiple Conflicting Phenotypes

ABSTRACT(p)ppGpp responds to nutrient limitation through a global change in gene regulation patterns to increase survival. The stringent response has been implicated in the virulence of several pathogenic bacterial species.Haemophilus ducreyi, the causative agent of chancroid, has homologs of bothrelAandspoT, which primarily synthesize and hydrolyze (p)ppGpp inEscherichia coli. We constructedrelAandrelA spoTdeletion mutants to assess the contribution of (p)ppGpp toH. ducreyipathogenesis. Both therelAsingle mutant and therelA spoTdouble mutant failed to synthesize (p)ppGpp, suggesting thatrelAis the primary synthetase of (p)ppGpp inH. ducreyi. Compared to the parent strain, the double mutant was partially attenuated for pustule formation in human volunteers. The double mutant had several phenotypes that favored attenuation, including increased sensitivity to oxidative stress. The increased sensitivity to oxidative stress could be complemented intrans. However, the double mutant also exhibited phenotypes that favored virulence. When grown to the mid-log phase, the double mutant was significantly more resistant than its parent to being taken up by human macrophages and exhibited increased transcription oflspB, which is involved in resistance to phagocytosis. Additionally, compared to the parent, the double mutant also exhibited prolonged survival in the stationary phase. InE. coli, overexpression of DksA compensates for the loss of (p)ppGpp; theH. ducreyidouble mutant expressed higher transcript levels ofdksAthan the parent strain. These data suggest that the partial attenuation of the double mutant is likely the net result of multiple conflicting phenotypes.

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Enhancing Terpene Yield from Sugars via Novel Routes to 1-Deoxy-d-Xylulose 5-Phosphate

Applied and Environmental Microbiology ◽

10.1128/aem.02920-14 ◽

2014 ◽

Vol 81 (1) ◽

pp. 130-138 ◽

Cited By ~ 30

Author(s):

James Kirby ◽

Minobu Nishimoto ◽

Ruthie W. N. Chow ◽

Edward E. K. Baidoo ◽

George Wang ◽

...

Keyword(s):

Bacterial Species ◽

Xylose Reductase ◽

Pentose Phosphate ◽

Terpene Biosynthesis ◽

Content Type ◽

E Coli ◽

Pathway Expression ◽

Terpene Synthesis ◽

Selection For

ABSTRACTTerpene synthesis in the majority of bacterial species, together with plant plastids, takes place via the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway. The first step of this pathway involves the condensation of pyruvate and glyceraldehyde 3-phosphate by DXP synthase (Dxs), with one-sixth of the carbon lost as CO2. A hypothetical novel route from a pentose phosphate to DXP (nDXP) could enable a more direct pathway from C5sugars to terpenes and also circumvent regulatory mechanisms that control Dxs, but there is no enzyme known that can convert a sugar into its 1-deoxy equivalent. Employing a selection for complementation of adxsdeletion inEscherichia coligrown on xylose as the sole carbon source, we uncovered two candidate nDXP genes. Complementation was achieved either via overexpression of the wild-typeE. coliyajOgene, annotated as a putative xylose reductase, or via various mutations in the nativeribBgene.In vitroanalysis performed with purified YajO and mutant RibB proteins revealed that DXP was synthesized in both cases from ribulose 5-phosphate (Ru5P). We demonstrate the utility of these genes for microbial terpene biosynthesis by engineering the DXP pathway inE. colifor production of the sesquiterpene bisabolene, a candidate biodiesel. To further improve flux into the pathway from Ru5P, nDXP enzymes were expressed as fusions to DXP reductase (Dxr), the second enzyme in the DXP pathway. Expression of a Dxr-RibB(G108S) fusion improved bisabolene titers more than 4-fold and alleviated accumulation of intracellular DXP.

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Combined Action of Human Commensal Bacteria and Amorphous Silica Nanoparticles on the Viability and Immune Responses of Dendritic Cells

Clinical and Vaccine Immunology ◽

10.1128/cvi.00178-17 ◽

2017 ◽

Vol 24 (10) ◽

Cited By ~ 3

Author(s):

Giulia Malachin ◽

Elisa Lubian ◽

Fabrizio Mancin ◽

Emanuele Papini ◽

Regina Tavano

Keyword(s):

Dendritic Cells ◽

Silica Nanoparticles ◽

Amorphous Silica ◽

Pathogenic Bacteria ◽

Bacterial Species ◽

Commensal Bacteria ◽

Content Type ◽

E Coli ◽

Synergistic Induction ◽

Ifn Γ

ABSTRACT Dendritic cells (DCs) regulate the host-microbe balance in the gut and skin, tissues likely exposed to nanoparticles (NPs) present in drugs, food, and cosmetics. We analyzed the viability and the activation of DCs incubated with extracellular media (EMs) obtained from cultures of commensal bacteria (Escherichia coli, Staphylococcus epidermidis) or pathogenic bacteria (Pseudomonas aeruginosa, Staphylococcus aureus) in the presence of amorphous silica nanoparticles (SiO2 NPs). EMs and NPs synergistically increased the levels of cytotoxicity and cytokine production, with different nanoparticle dose-response characteristics being found, depending on the bacterial species. E. coli and S. epidermidis EMs plus NPs at nontoxic doses stimulated the secretion of interleukin-1β (IL-1β), IL-12, IL-10, and IL-6, while E. coli and S. epidermidis EMs plus NPs at toxic doses stimulated the secretion of gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), IL-4, and IL-5. On the contrary, S. aureus and P. aeruginosa EMs induced cytokines only when they were combined with NPs at toxic concentrations. The induction of maturation markers (CD86, CD80, CD83, intercellular adhesion molecule 1, and major histocompatibility complex class II) by commensal bacteria but not by pathogenic ones was improved in the presence of noncytotoxic SiO2 NP doses. DCs consistently supported the proliferation and differentiation of CD4+ and CD8+ T cells secreting IFN-γ and IL-17A. The synergistic induction of CD86 was due to nonprotein molecules present in the EMs from all bacteria tested. At variance with this finding, the synergistic induction of IL-1β was prevalently mediated by proteins in the case of E. coli EMs and by nonproteins in the case of S. epidermidis EMs. A bacterial costimulus did not act on DCs after adsorption on SiO2 NPs but rather acted as an independent agonist. The inflammatory and immune actions of DCs stimulated by commensal bacterial agonists might be altered by the simultaneous exposure to engineered or environmental NPs.

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Escherichia coli Clonobiome: Assessing the Strain Diversity in Feces and Urine by Deep Amplicon Sequencing

Applied and Environmental Microbiology ◽

10.1128/aem.01866-19 ◽

2019 ◽

Vol 85 (23) ◽

Author(s):

Sofiya G. Shevchenko ◽

Matthew Radey ◽

Veronika Tchesnokova ◽

Dagmara Kisiela ◽

Evgeni V. Sokurenko

Keyword(s):

Bacterial Species ◽

Clonal Diversity ◽

Amplicon Sequencing ◽

Multidrug Resistant ◽

Full Understanding ◽

Highly Pathogenic ◽

Content Type ◽

E Coli ◽

Strain Diversity ◽

Healthy Carriers

ABSTRACT While microbiome studies have focused on diversity at the species level or higher, bacterial species in microbiomes are represented by different, often multiple, strains. These strains could be clonally and phenotypically very different, making assessment of strain content vital to a full understanding of microbiome function. This is especially important with respect to antibiotic-resistant strains, the clonal spread of which may be dependent on competition between them and susceptible strains from the same species. The pandemic, multidrug-resistant, and highly pathogenic Escherichia coli subclone ST131-H30 (H30) is of special interest, as it has already been found persisting in the gut and bladder in healthy people. In order to rapidly assess E. coli clonal diversity, we developed a novel method based on deep sequencing of two loci used for sequence typing, along with an algorithm for analysis of the resulting data. Using this method, we assessed fecal and urinary samples from healthy women carrying H30 and were able to uncover considerable diversity, including strains with frequencies at <1% of the E. coli population. We also found that, even in the absence of antibiotic use, H30 could completely dominate the gut and, especially, urine of healthy carriers. Our study offers a novel tool for assessing a species’ clonal diversity (clonobiome) within the microbiome, which could be useful in studying the population structure and dynamics of multidrug-resistant and/or highly pathogenic strains in their natural environments. IMPORTANCE Bacterial species in the microbiome are often represented by multiple genetically and phenotypically different strains, making insight into subspecies diversity critical to a full understanding of the microbiome, especially with respect to opportunistic pathogens. However, methods allowing efficient high-throughput clonal typing are not currently available. This study combines a conventional E. coli typing method with deep amplicon sequencing to allow analysis of many samples concurrently. While our method was developed for E. coli, it may be adapted for other species, allowing microbiome researchers to assess clonal strain diversity in natural samples. Since assessment of subspecies diversity is particularly important for understanding the spread of antibiotic resistance, we applied our method to the study of a pandemic multidrug-resistant E. coli clone. The results we present suggest that this clone could be highly competitive in healthy carriers and that the mechanisms of colonization by such clones need to be studied.

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A Stringent Analysis of Polyphosphate Dynamics inEscherichia coli

Journal of Bacteriology ◽

10.1128/jb.00070-19 ◽

2019 ◽

Vol 201 (9) ◽

Author(s):

Michael Downey

Keyword(s):

Escherichia Coli ◽

Infection Control ◽

Bacterial Species ◽

Stringent Response ◽

Industrial Applications ◽

Bacterial Cells ◽

Inescherichia Coli ◽

Content Type ◽

Polyphosphate Accumulation ◽

Inorganic Phosphates

ABSTRACTDuring stress, bacterial cells activate a conserved pathway called the stringent response that promotes survival. Polyphosphates are long chains of inorganic phosphates that modulate this response in diverse bacterial species. In this issue, Michael J. Gray provides an important correction to the model of how polyphosphate accumulation is regulated during the stringent response inEscherichia coli(M. J. Gray, J. Bacteriol, 201:e00664-18, 2019,https://doi.org/10.1128/JB.00664-18). With other recent publications, this study provides a revised framework for understanding how bacterial polyphosphate dynamics might be exploited in infection control and industrial applications.

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Evolutionary Analysis Points to Divergent Physiological Roles of Type 1 Fimbriae in Salmonella and Escherichia coli

mBio ◽

10.1128/mbio.00625-12 ◽

2013 ◽

Vol 4 (2) ◽

Cited By ~ 6

Author(s):

Dagmara I. Kisiela ◽

Sujay Chattopadhyay ◽

Veronika Tchesnokova ◽

Sandip Paul ◽

Scott J. Weissman ◽

...

Keyword(s):

Bacterial Species ◽

Structural Diversity ◽

Evolutionary Analysis ◽

Content Type ◽

E Coli ◽

Type 1 Fimbriae ◽

Gene Shuffling ◽

Immune Pressure ◽

High Level

ABSTRACTSalmonellaandEscherichia colimannose-binding type 1 fimbriae exhibit highly similar receptor specificities, morphologies, and mechanisms of assembly but are nonorthologous in nature, i.e., not closely related evolutionarily. Their operons differ in chromosomal location, gene arrangement, and regulatory components. In the current study, we performed a comparative genetic and structural analysis of the major structural subunit, FimA, fromSalmonellaandE. coliand found that FimA pilins undergo diverse evolutionary adaptation in the different species. Whereas theE. coli fimAlocus is characterized by high allelic diversity, frequent intragenic recombination, and horizontal movement,Salmonella fimAshows structural diversity that is more than 5-fold lower without strong evidence of gene shuffling or homologous recombination. In contrast toSalmonellaFimA, the amino acid substitutions in theE. colipilin heavily target the protein regions that are predicted to be exposed on the external surface of fimbriae. Altogether, our results suggest thatE. coli, but notSalmonella, type 1 fimbriae display a high level of structural diversity consistent with a strong selection for antigenic variation under immune pressure. Thus, type 1 fimbriae in these closely related bacterial species appear to function in distinctly different physiological environments.IMPORTANCEE. coliandSalmonellaare enteric bacteria that are closely related from an evolutionary perspective. They are both notorious human pathogens, though with somewhat distinct ecologies and virulence mechanisms. Type 1 fimbriae are rod-shaped surface appendages found in mostE. coliandSalmonellaisolates. In both species, they mediate bacterial adhesion to mannose receptors on host cells and share essentially the same morphology and assembly mechanisms. Here we show that despite the strong resemblances in function and structure, they are exposed to very different natural selection environments. Sequence analysis indicates thatE. coli, but notSalmonella, fimbriae are subjected to strong immune pressure, resulting in a high level of major fimbrial protein gene shuffling and interbacterial transfer. Thus, evolutionary analysis tools can provide evidence of divergent physiological roles of functionally similar traits in different bacterial species.

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RpoS Contributes to Phagocyte Oxidase-Mediated Stress Resistance during Urinary Tract Infection by Escherichia coli CFT073

mBio ◽

10.1128/mbio.00023-13 ◽

2013 ◽

Vol 4 (1) ◽

Cited By ~ 26

Author(s):

Andrew J. Hryckowian ◽

Rodney A. Welch

Keyword(s):

Oxidative Stress ◽

Urinary Tract ◽

Wild Type ◽

Upec Strain ◽

Content Type ◽

General Stress Response ◽

E Coli ◽

Phagocyte Oxidase ◽

Strain Cft073 ◽

K 12

ABSTRACTUropathogenicEscherichia coli(UPEC) is the most common causative agent of community-acquired urinary tract infection (UTI). In order to cause UTI, UPEC must endure stresses ranging from nutrient limitation to host immune components. RpoS (σS), the general stress response sigma factor, directs gene expression under a variety of inhibitory conditions. Our study ofrpoSin UPEC strain CFT073 began after we discovered anrpoS-frameshift mutation in one of our laboratory stocks of “wild-type” CFT073. We demonstrate that anrpoS-deletion mutation in CFT073 leads to a colonization defect during UTI of CBA/J mice at 48 hours postinfection (hpi). There is no difference between the growth rates of CFT073 and CFT073rpoSin urine. This indicates thatrpoSis needed for replication and survival in the host rather than being needed to address limitations imposed by urine nutrients. Consistent with previous observations inE. coliK-12, CFT073rpoSis more sensitive to oxidative stress than the wild type. We demonstrate that peroxide levels are elevated in voided urine from CFT073-infected mice compared to urine from mock-infected mice, which supports the notion that oxidative stress is generated by the host in response to UPEC. In mice that lack phagocyte oxidase, the enzyme complex expressed by phagocytes that produces superoxide, the competitive defect of CFT073rpoSin bladder colonization is lost. These results demonstrate that σSis important for UPEC survival under conditions of phagocyte oxidase-generated stress during UTI. Though σSaffects the pathogenesis of other bacterial species, this is the first work that directly implicates σSas important for UPEC pathogenesis.IMPORTANCEUPEC must cope with a variety of stressful conditions in the urinary tract during infection. RpoS (σS), the general stress response sigma factor, is known to direct the expression of many genes under a variety of stressful conditions in laboratory-adaptedE. coliK-12. Here, we show that σSis needed by the model UPEC strain CFT073 to cope with oxidative stress provided by phagocytes during infection. These findings represent the first report that implicates σSin the fitness of UPEC during infection and support the idea of the need for a better understanding of the effects of this global regulator of gene expression during UTI.

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Carbon Storage Regulator A Contributes to the Virulence of Haemophilus ducreyi in Humans by Multiple Mechanisms

Infection and Immunity ◽

10.1128/iai.01239-12 ◽

2012 ◽

Vol 81 (2) ◽

pp. 608-617 ◽

Cited By ~ 13

Author(s):

Dharanesh Gangaiah ◽

Wei Li ◽

Kate R. Fortney ◽

Diane M. Janowicz ◽

Sheila Ellinger ◽

...

Keyword(s):

Oxidative Stress ◽

Stationary Phase ◽

Carbon Storage ◽

Stress Responses ◽

Haemophilus Ducreyi ◽

Content Type ◽

Enhanced Sensitivity ◽

Significant Survival ◽

Carbon Storage Regulator ◽

Mutant Phenotypes

ABSTRACTThe carbon storage regulator A (CsrA) controls a wide variety of bacterial processes, including metabolism, adherence, stress responses, and virulence.Haemophilus ducreyi, the causative agent of chancroid, harbors a homolog ofcsrA. Here, we generated an unmarked, in-frame deletion mutant ofcsrAto assess its contribution toH. ducreyipathogenesis. In human inoculation experiments, thecsrAmutant was partially attenuated for pustule formation compared to its parent. Deletion ofcsrAresulted in decreased adherence ofH. ducreyito human foreskin fibroblasts (HFF); Flp1 and Flp2, the determinants ofH. ducreyiadherence to HFF cells, were downregulated in thecsrAmutant. Compared to its parent, thecsrAmutant had a significantly reduced ability to tolerate oxidative stress and heat shock. The enhanced sensitivity of the mutant to oxidative stress was more pronounced in bacteria grown to stationary phase compared to that in bacteria grown to mid-log phase. ThecsrAmutant also had a significant survival defect within human macrophages when the bacteria were grown to stationary phase but not to mid-log phase. Complementation intranspartially or fully restored the mutant phenotypes. These data suggest that CsrA contributes to virulence by multiple mechanisms and that these contributions may be more profound in bacterial cell populations that are not rapidly dividing in the human host.

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A DltA Mutant of Haemophilus ducreyi Is Partially Attenuated in Its Ability To Cause Pustules in Human Volunteers

Infection and Immunity ◽

10.1128/iai.74.2.1394-1397.2006 ◽

2006 ◽

Vol 74 (2) ◽

pp. 1394-1397 ◽

Cited By ~ 14

Author(s):

Diane Janowicz ◽

Isabelle Leduc ◽

Kate R. Fortney ◽

Barry P. Katz ◽

Christopher Elkins ◽

...

Keyword(s):

Confidence Interval ◽

Human Serum ◽

Parent Strain ◽

Outer Membrane Proteins ◽

The Other ◽

Haemophilus Ducreyi ◽

Serum Resistance ◽

Human Volunteers ◽

Serum Killing ◽

Normal Human

ABSTRACT Haemophilus ducreyi produces two outer membrane proteins, called DltA (H. ducreyi lectin A) and DsrA (H. ducreyi serum resistance A), that contribute to the ability of the organism to evade complement-mediated serum killing. In contrast to their isogenic parent strain, 35000HP, the DsrA mutant FX517 exhibits 0% survival in 50% normal human serum and the DltA mutant FX533 exhibits 23% survival. Compared to 35000HP, FX517 does not cause pustule formation in human volunteers. To test whether DltA was required for virulence in humans, seven volunteers were experimentally infected with 35000HP and FX533. Four subjects were inoculated with fixed doses of 35000HP (101 CFU or 130 CFU) at three sites on one arm and escalating doses of FX533 (range, 46 CFU to 915 CFU) at three sites on the other arm. Pustules only developed at mutant-injected sites at doses nearly twofold higher than that of the parent, suggesting that FX533 was partially attenuated. Three subjects were inoculated with similar doses of the parent (67 CFU) and mutant (104 CFU) at three sites. Pustules formed at five of nine parent sites and one of nine mutant sites. Overall, the papule and pustule formation rates for 35000HP and FX533 were similar for the trial. However, for the five subjects who received similar doses of the parent and mutant, pustules developed at 7 of 15 sites (46.7%; 95% confidence interval [CI], 16.9% to 76.5%) inoculated with the parent and at 1 of 15 (6.7%; 95% CI, 0.1% to 18.4%) sites inoculated with the mutant (P = 0.043). We concluded that the DltA mutant was attenuated in its ability to cause disease at doses similar to that of the parent.

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Antimicrobial Properties of a Chitosan Dextran-Based Hydrogel for Surgical Use

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05463-11 ◽

2011 ◽

Vol 56 (1) ◽

pp. 280-287 ◽

Cited By ~ 80

Author(s):

Manal A. Aziz ◽

Jaydee D. Cabral ◽

Heather J. L. Brooks ◽

Stephen C. Moratti ◽

Lyall R. Hanton

Keyword(s):

Electron Microscopy ◽

Transmission Electron Microscopy ◽

Bacterial Species ◽

Antimicrobial Properties ◽

Sinus Surgery ◽

Peptide Bonds ◽

Content Type ◽

E Coli ◽

Transmission Electron

ABSTRACTA chitosan dextran-based (CD) hydrogel, developed for use in endoscopic sinus surgery, was tested for antimicrobial activityin vitroagainst a range of pathogenic microorganisms. The microdilution technique was used to determine minimum inhibitory, minimum bactericidal, and minimum fungicidal concentrations. In addition, the time-kill efficacy of CD hydrogel was determined for two bacterial species. Scanning and transmission electron microscopy were carried out to elucidate the antimicrobial mechanism of this compound. CD hydrogel was found to be effective againstStaphylococcus aureus,Streptococcus pyogenes,Escherichia coli, andClostridium perfringensat its surgical concentration of 50,000 mg/liter. Minimum bactericidal concentrations ranged from 2,000 to 50,000 mg/liter. Dextran aldehyde (DA) was found to be the antimicrobial component of the CD hydrogel with MBC ranging from 2,000 to 32,000 mg/liter.S. aureusappeared to be killed at a slightly faster rate thanE. coli. Candida albicansandPseudomonas aeruginosawere more resistant to CD hydrogel and DA. Scanning and transmission electron microscopy ofE. coliandS. aureusincubated with CD hydrogel and DA alone revealed morphological damage, disrupted cell walls, and loss of cytosolic contents, compatible with the proposed mode of action involving binding to cell wall proteins and disruption of peptide bonds. Motility and chemotaxis tests showedE. colito be inhibited when incubated with DA. The antibacterial activity of CD hydrogel may make it a useful postsurgical aid at other body sites, especially where there is a risk of Gram-positive infections.

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Identification of Escherichia coli and Shigella Species from Whole-Genome Sequences

Journal of Clinical Microbiology ◽

10.1128/jcm.01790-16 ◽

2016 ◽

Vol 55 (2) ◽

pp. 616-623 ◽

Cited By ~ 49

Author(s):

Marie A. Chattaway ◽

Ulf Schaefer ◽

Rediat Tewolde ◽

Timothy J. Dallman ◽

Claire Jenkins

Keyword(s):

Escherichia Coli ◽

Clonal Complex ◽

Shigella Flexneri ◽

Bacterial Species ◽

Whole Genome ◽

Content Type ◽

E Coli ◽

Shigella Species ◽

Close Phylogenetic Relationship ◽

Initial Identification

ABSTRACTEscherichia coliandShigellaspecies are closely related and genetically constitute the same species. Differentiating between these two pathogens and accurately identifying the four species ofShigellaare therefore challenging. The organism-specific bioinformatics whole-genome sequencing (WGS) typing pipelines at Public Health England are dependent on the initial identification of the bacterial species by use of a kmer-based approach. Of the 1,982Escherichia coliandShigellasp. isolates analyzed in this study, 1,957 (98.4%) had concordant results by both traditional biochemistry and serology (TB&S) and the kmer identification (ID) derived from the WGS data. Of the 25 mismatches identified, 10 were enteroinvasiveE. coliisolates that were misidentified asShigella flexneriorS. boydiiby the kmer ID, and 8 wereS. flexneriisolates misidentified by TB&S asS. boydiidue to nonfunctionalS. flexneriO antigen biosynthesis genes. Analysis of the population structure based on multilocus sequence typing (MLST) data derived from the WGS data showed that the remaining discrepant results belonged to clonal complex 288 (CC288), comprising bothS. boydiiandS. dysenteriaestrains. Mismatches between the TB&S and kmer ID results were explained by the close phylogenetic relationship between the two species and were resolved with reference to the MLST data.Shigellacan be differentiated fromE. coliand accurately identified to the species level by use of kmer comparisons and MLST. Analysis of the WGS data provided explanations for the discordant results between TB&S and WGS data, revealed the true phylogenetic relationships between different species ofShigella, and identified emerging pathoadapted lineages.

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