Isogenic Wild-type Strain Research Articles

A New Bacterial Chassis for Enhanced Surface Display of Recombinant Proteins.

Bacterial surface display is a valuable biotechnology technique for presenting proteins and molecules on the outer surface of bacterial cells. However, it has limitations, including potential toxicity to host bacteria and variability in display efficiency. To address these issues, we investigated the removal of abundant non-essential outer membrane proteins (OMPs) in E. coli as a new strategy to improve the surface display of recombinant proteins. We targeted OmpA, a highly prevalent OMP in E. coli, using the lambda red method. We successfully knocked out ompA in two E. coli strains, K-12 MG1655 and E. coli BL-21, which have broad research and therapeutic applications. We then combined ompA knockout strains and two OMPs with three therapeutic proteins including an anti-toxin enzyme (ClbS), interleukin 18 (IL-18) for activating cytotoxic T cells and an anti- CTLA4 nanobody (αCTLA4) for immune checkpoint blockade. A total of six different display constructs were tested for their display levels by flow cytometry, showing that the ompA knockout strains increased the percentage as well as the levels of display in bacteria compared to those of isogenic wild-type strains. By removing non-essential, highly abundant surface proteins, we develop an efficient platform for displaying enzymes and antibodies, with potential industrial and therapeutic applications. Additionally, the enhanced therapeutic efficacy opens possibilities for live bacteria-based therapeutics, expanding the technology's relevance in the field. The online version contains supplementary material available at 10.1007/s12195-024-00819-w.

TFIIB-Termination Factor Interaction Affects Termination of Transcription on Genome-Wide Scale.

Apart from its well-established role in the initiation of transcription, the general transcription factor TFIIB has been implicated in the termination step as well. The ubiquity of TFIIB involvement in termination as well as mechanistic details of its termination function, however, remain largely unexplored. Using GRO-seq analyses, we compared the terminator readthrough phenotype in the sua7-1 mutant (TFIIBsua7-1) and the isogenic wild type (TFIIBWT) strains. Approximately 74% of genes analyzed exhibited a 2-3-fold increase in readthrough of the poly(A)-termination signal in the TFIIBsua7-1 mutant compared to TFIIBWT cells. To understand the mechanistic basis of TFIIB's role in termination, we performed the mass spectrometry of TFIIB-affinity purified from chromatin and soluble cellular fractions-from TFIIBsua7-1 and TFIIBWT cells. TFIIB purified from the chromatin fraction of TFIIBWT cells exhibited significant enrichment of CF1A and Rat1 termination complexes. There was, however, a drastic decrease in TFIIB interaction with CF1A and Rat1 complexes in the TFIIBsua7-1 mutant. ChIP assays revealed about a 90% decline in the recruitment of termination factors in the TFIIBsua7-1 mutant compared to wild type cells. The overall conclusion of these results is that TFIIB affects the termination of transcription on a genome-wide scale, and the TFIIB-termination factor interaction plays a crucial role in the process.

Genome-wide analysis of TFIIB's role in termination of transcription.

This study provides evidence that the role of TFIIB extends beyond initiation to include the termination step of transcription. Using GRO-seq analyses, we compared terminator readthrough phenotype in sua7-1 mutant (TFIIB sua7-1 ) and the isogenic wild type (TFIIB WT ) strains. Approximately 74% of genes analyzed exhibited a 2-3-fold increase in readthrough of the poly(A)-termination signal in the TFIIB sua7-1 mutant compared to TFIIB WT cells. Mass spectrometry of affinity purified TFIIB from chromatin fraction found TFIIB exhibiting interaction with CF1A and Rat1 termination complexes in TFIIB WT cells. There was, however, a drastic decrease in TFIIB interaction with CF1A and Rat1 termination complexes in the TFIIB sua7-1 mutant. ChIP assays revealed about 90% decline in recruitment of termination factors in TFIIB sua7-1 mutant compared to wild type cells. The overall conclusion of these results is that TFIIB affects termination of transcription on a genome-wide scale, and TFIIB-termination factor interaction may play a crucial role in the process.

The serine-rich repeat glycoprotein Srr2 mediates Streptococcus agalactiae interaction with host fibronectin

BackgroundGroup B Streptococcus (GBS) is a commensal of healthy adults and an important pathogen in newborns, the elderly and immunocompromised individuals. GBS displays several virulence factors that promote colonisation and host infection, including the ST-17 strain-specific adhesin Srr2, previously characterised for its binding to fibrinogen. Another common target for bacterial adhesins and for host colonization is fibronectin, a multi-domain glycoprotein found ubiquitously in body fluids, in the extracellular matrix and on the surface of cells.ResultsIn this study, fibronectin was identified as a novel ligand for the Srr2 adhesin of GBS. A derivative of the ST-17 strain BM110 overexpressing the srr2 gene showed an increased ability to bind fibrinogen and fibronectin, compared to the isogenic wild-type strain. Conversely, the deletion of srr2 impaired bacterial adhesion to both ligands. ELISA assays and surface plasmon resonance studies using the recombinant binding region (BR) form of Srr2 confirmed a direct interaction with fibronectin with an estimated Kd of 92 nM. Srr2-BR variants defective in fibrinogen binding also exhibited no interaction with fibronectin, suggesting that Srr2 binds this ligand through the dock-lock-latch mechanism, previously described for fibrinogen binding. The fibronectin site responsible for recombinant Srr2-BR binding was identified and localised in the central cell-binding domain of the protein. Finally, in the presence of fibronectin, the ability of a Δsrr2 mutant to adhere to human cervico-vaginal epithelial cells was significantly lower than that of the wild-type strain.ConclusionBy combining genetic and biochemical approaches, we demonstrate a new role for Srr2, namely interacting with fibronectin. We characterised the molecular mechanism of this interaction and demonstrated that it plays a role in promoting the adhesion of GBS to human cervico-vaginal epithelial cells, further substantiating the role of Srr2 as a factor responsible for the hypervirulence of GBS ST-17 strains. The discovery of the previously undescribed interaction between Srr2 and fibronectin establishes this adhesin as a key factor for GBS colonisation of host tissues.

Genome-wide analysis of TFIIB's role in termination of transcription.

Apart from its well-established role in initiation of transcription, the general transcription factor TFIIB has been implicated in the termination step as well. The ubiquity of TFIIB involvement in termination as well as mechanistic details of its termination function, however, remains largely unexplored. To determine the prevalence of TFIIB's role in termination, we performed GRO-seq analyses in sua7-1 mutant (TFIIB sua7-1 ) and the isogenic wild type (TFIIB WT ) strains of yeast. Almost a three-fold increase in readthrough of the poly(A)-termination signal was observed in TFIIB sua7-1 mutant compared to the TFIIB WT cells. Of all genes analyzed in this study, nearly 74% genes exhibited a statistically significant increase in terminator readthrough in the mutant. To gain an understanding of the mechanistic basis of TFIIB involvement in termination, we performed mass spectrometry of TFIIB, affinity purified from chromatin and soluble cellular fractions, from TFIIB sua7-1 and TFIIB WT cells. TFIIB purified from the chromatin fraction of TFIIB WT cells exhibited significant enrichment of CF1A and Rat1 termination complexes. There was, however, a drastic decrease in TFIIB interaction with both CF1A and Rat1 termination complexes in TFIIB sua7-1 mutant. ChIP assay revealed that the recruitment of Pta1 subunit of CPF complex, Rna15 subunit of CF1 complex and Rat1 subunit of Rat1 complex registered nearly 90% decline in the mutant over wild type cells. The overall conclusion of these results is that TFIIB affects termination of transcription on a genome-wide scale, and TFIIB-termination factor interaction may play a crucial role in the process.

Pseudomonas aeruginosa Phosphate Transporter PitA (PA4292) Controls Susceptibility to Aminoglycoside Antibiotics by Regulating the Proton Motive Force.

Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium that causes nosocomial infections in immunocompromised patients. β-lactam and aminoglycoside antibiotics are commonly used in the treatment of P. aeruginosa infections. Previously, we found that mutation in a PA4292 gene increases bacterial resistance to β-lactam antibiotics. In this study, we demonstrated that mutation in PA4292 increases bacterial susceptibility to aminoglycoside antibiotics. We further found enhanced uptake of tobramycin by the ΔPA4292 mutant, which might be due to an increase of proton motive force (PMF). Sequence analysis revealed PA4292 is homologous to the Escherichia coli phosphate transporter PitA. Mutation of PA4292 indeed reduces intracellular phosphate concentration. We thus named PA4292 as pitA. Although the PMF is enhanced in the ΔpitA mutant, the intracellular ATP concentration is lower than that in the isogenic wild-type strain PA14, which might be due to lack of the ATP synthesis substrate phosphate. Overexpression of the phosphate transporter complex genes pstSCAB in the ΔpitA mutant restores the intracellular phosphate concentration, PMF, ATP synthesis, and aminoglycosides resistance. In addition, growth of wild-type PA14 in a low-phosphate medium resulted in higher PMF and aminoglycoside susceptibility compared to cells grown in a high-phosphate medium. Overall, our results demonstrate the roles of PitA in phosphate transportation and reveal the relationship between intracellular phosphate and aminoglycoside susceptibility.

Effect of a single amino acid substitution G98D in a ribosome-associated essential GTPase, CgtA, on the growth and morphology of Vibrio cholerae

CgtA, a highly conserved 50S ribosome-associated essential GTPase, acts as a repressor of the stringent stress response under nutrient-rich growth conditions to suppress basal levels of the alarmone ppGpp in V. cholerae. To further explore the in vivo functionality of CgtA, we introduced an amino acid substitution, i.e., Gly98Asp, in a conserved glycine residue in the N-terminal domain. The constructed V. cholerae mutant was designated CgtA(G98D). Comparison of cell sizes of the CgtA(G98D)mutant with its isogenic wild-type (Wt) strain N16961 under different phases of growth by Transmission Electron Microscopy (TEM) and statistical analysis suggests that CgtA may control the cell size of V. cholerae. The cell length is significantly reduced, corresponding to the delayed growth in the mid-logarithmic phase. The differences in the cell length of CgtA(G98D) and Wt are indistinguishable in the late logarithmic phase. During the stationary phase, marked by higher OD600, a sub-population of CgtA(G98D) cells outnumbered the Wt cells lengthwise. CgtA(G98D) cells appeared slenderer than Wt cells with significantly reduced cell width. However, the centerline curvature is preserved in CgtA(G98D) cells. We propose that in addition to its multitude of intracellular roles, CgtA may influence the cell size of V. cholerae.

The rnc Gene Regulates the Microstructure of Exopolysaccharide in the Biofilm of Streptococcus mutans through the β-Monosaccharides

Streptococcus mutans is known as the crucial pathogen of human dental caries, owing to its contribution to the biofilm development via the capacity of synthesizing exopolysaccharide (EPS), which mainly compose of α-glycosidic bond and β-glycosidic bond. β-glycosidic bond is less flexible than α-glycosidic bond because of differences between their configurational properties. Previous studies have shown that the rnc gene is implicated in the EPS formation and the cariogenicity of S. mutans. However, the effects of rnc on the microstructure of EPS have been not well-understood yet. Here, we further investigated how the rnc gene worked to modulate microstructural properties of the extracellular polysaccharide of S. mutans using glycomics methods. The gas chromatography-mass spectrometer showed that the proportion of glucose was decreased in water-soluble EPS and galactose was absent in water-insoluble EPS from the S. mutans rnc-deficient strain (Smurnc), compared with the isogenic wild-type strain (UA159). The composition of functional groups and the displacement of hydrogen bond were analyzed by infrared radiation and 1H nuclear magnetic resonance, respectively. In addition, phenotypic modulation of the biofilm matrix was assessed by microscopy. We found that the EPS of UA159 and the rnc overexpression strain (Smurnc+) mainly consisted of β-glycosidic bonds. Conversely, the EPS of Smurnc were made up of mostly α-glycosidic bonds, leading to the attenuation of biofilm biomass and bacterial adhesion. Furthermore, the existence of β-glycosidic bond was verified by enzyme digestion. Collectively, the rnc gene modulates the conversion of β-glycosidic bonds, which may play important roles in regulating the micromolecule structure of the EPS matrix, thus affecting the characteristics of S. mutans biofilm. These data illustrate that β-glycosidic bonds mediated by rnc may be potential targets for the prevention and treatment of dental caries.

The DmsABC Sulfoxide Reductase Supports Virulence in Non-typeable Haemophilus influenzae.

Although molybdenum-containing enzymes are well-established as having a key role in bacterial respiration, it is increasingly recognized that some may also support bacterial virulence. Here, we show that DmsABC, a putative dimethylsulfoxide (DMSO) reductase, is required for fitness of the respiratory pathogen Haemophilus influenzae (Hi) in different models of infection. Expression of the dmsABC operon increased with decreasing oxygen availability, but despite this, a Hi2019ΔdmsA strain did not show any defects in anaerobic growth on chemically defined medium (CDM), and viability was also unaffected. Although Hi2019ΔdmsA exhibited increased biofilm formation in vitro and greater resistance to hypochlorite killing compared to the isogenic wild-type strain, its survival in contact with primary human neutrophils, in infections of cultured tissue cells, or in a mouse model of lung infection was reduced compared to Hi2019WT. The tissue cell infection model revealed a two-fold decrease in intracellular survival, while in the mouse model of lung infection Hi2019ΔdmsA was strongly attenuated and below detection levels at 48 h post-inoculation. While Hi2019WT was recovered in approximately equal numbers from bronchoalveolar lavage fluid (BALF) and lung tissue, survival of Hi2019ΔdmsA was reduced in lung tissue compared to BALF samples, indicating that Hi2019ΔdmsA had reduced access to or survival in the intracellular niche. Our data clearly indicate for the first time a role for DmsABC in H. influenzae infection and that the conditions under which DmsABC is required in this bacterium are closely linked to interactions with the host.

Varied Contribution of Phospholipid Shedding From Membrane to Daptomycin Tolerance in Staphylococcus aureus.

It has been suggested that daptomycin can be inactivated by lipids released by Staphylococcus aureus and that this effect is antagonized by phenol soluble modulins (PSMs), which bind to the shed lipids. PSM production is regulated by the Agr system, and others have shown that loss of the Agr function enhances S. aureus survival in the presence of daptomycin. Here we assessed the impact of Agr function on daptomycin activity and lipid metabolism under various conditions. Daptomycin activity was evaluated against three sets of isogenic strain series with wild-type or dysfunctional Agr using static daptomycin time-kills over 24 h and against one strain pair using in vitro pharmacokinetic/pharmacodynamic (PK/PD) models simulating clinical daptomycin exposure for 48 h. We performed comprehensive lipidomics on bacterial membranes and the spent media to correlate lipid shedding with survival. In static time-kill experiments, two agr-deficient strains (SH1000- and USA300 LAC ΔagrA) showed improved survival for 8 h compared with their corresponding wild-type strains as seen in previous studies, but this difference did not persist for 24 h. However, four other agr-deficient strains (SH1001 and JE2 agr KOs) did not demonstrate improved survival compared to isogenic wild-type strains at any time in the time-kills. Lipidomics analysis of SH1000, SH1001, and SH1000- strains showed daptomycin exposure increased lipid shedding compared to growth controls in all strains with phosphatidylglycerols (PGs), lysylPGs and cardiolipins predominating. In the cell pellets, PGs and lysylPGs decreased but cardiolipins were unchanged with daptomycin exposure. The shed lipid profiles in SH1001 and SH1000- were similar, suggesting that the inability to resist daptomycin by SH1001 was not because of differences in lipid shedding. In the PK/PD model, the agr mutant SH1000- strain did not show improved survival relative to SH1000 either. In conclusion, inactivation of daptomycin by shed lipids may be dependent on genetic background, the specific agr mutations, or the techniques used to generate these KOs rather than the overall function of the Agr system, and its contribution to daptomycin tolerance seems to be varied, transient, and growth-condition dependent.

The serine protease HtrA plays a key role in heat-induced dispersal of pneumococcal biofilms

Streptococcus pneumoniae (the pneumococcus) colonizes the human nasopharynx by forming multicellular biofilms. Due to the high level of asymptomatic carriage, transition to infections, such as otitis media, pneumonia, sepsis, and meningitis, occurs often enough that the pneumococcus remains a major cause of disease and death globally. Virus infection and virus-induced responses, such as increased temperature (fever), trigger release of virulent bacteria from colonizing biofilms. The exact mechanisms involved in pneumococcal egress during biofilm dispersal remain unknown, although we hypothesize that disruption of the biofilm matrix encasing the bacteria is necessary. Here, we utilized established in vitro biofilm dispersal models to investigate the involvement of proteases in bacterial egress from pneumococcal biofilms. We demonstrate the importance of protease activity, both through increased bacterial release following addition of proteases and reduced heat-induced biofilm dispersal in the presence of protease inhibitors. We identify a key role for the surface-exposed serine protease HtrA, but not PrtA, in heat-induced biofilm dispersal. Bacterial release from htrA-negative biofilms was significantly reduced compared to wild-type isogenic strains but was restored and increased above wild-type levels following addition of recombinant HtrA. Understanding the specific mechanisms involved in bacterial egress may provide novel targets for future strategies aimed to specifically interfere with disease progression without disturbing nasopharyngeal biofilm colonization.

Distribution of fluoroquinolones in the two aqueous compartments of Escherichia coli

The double-layered cell envelope of Gram-negative bacteria and active drug efflux present a formidable barrier for antimicrobial compounds to penetrate. Fluoroquinolones are among the few classes of antimicrobials that are clinically useful in the treatment of Gram-negative bacterial infection. Previous studies on fluoroquinolone accumulation measured total bacteria associated compounds, rather than the cytoplasmic accumulation. Fluoroquinolones target the type II topoisomerases in the cytoplasm. Thus, the cytoplasmic accumulation is expected to be more relevant to the potency of the drugs. Here, we fractionated and measured the concentration of nine fluoroquinolone compounds in the periplasm and the cytoplasm of two strains of E. coli cells, a parent strain and its isogenic efflux-deficient tolC knockout strain. The potency of the drugs was determined using the minimum inhibitory concentration (MIC) assay. We found that all fluoroquinolones tested accumulated at much higher concentrations in the periplasm than in the cytoplasm. The periplasmic concentrations were 2–15 folds higher than the cytoplasmic concentration, while the actual distribution ratio varies drastically among the compounds tested. Good correlation between the MIC and the cytoplasmic accumulation, but not whole cell accumulation, was observed using a pair of isogenic wild type and drug-efflux deficient strains.

Characterization of a Chlamydomonas reinhardtii mutant strain with tolerance to low nitrogen and increased growth and biomass under nitrogen stress

Nitrogen is a common limiting nutrient for growth and biomass production in algae and plants. Understanding uptake, assimilation, and regulation of nitrogen metabolism to maximize biomass production with minimal nitrogen input is of great interest environmentally, for large-scale algal culturing, and for crop plants. We have characterized a new strain of Chlamydomonas reinhardtii that has a tolerance to low nitrogen (TLN) with higher growth under nitrogen stress compared to an isogenic wild-type (WT) strain. When grown in reduced ammonium the TLN1 strain has a significantly higher cell density, viable cell count, and biomass than WT. In 0.75 mM ammonium (one tenth the normal concentration in TAP medium) TLN1 has 30–40% more growth and ~55% higher biomass than WT. In 1.8 mM ammonium (one fourth the normal concentration in TAP) TLN1 has ~17% more growth and ~28% higher biomass than WT grown in normal high ammonium (7.5 mM), suggesting an altered regulation of nitrogen metabolism when under nitrogen stress. TLN1 also showed higher growth under nitrogen stress with urea and arginine as alternative sources of nitrogen. Genetic analysis indicates that the tln1 trait is inherited and due to a loss-of-function recessive mutation. In addition to the TLN1 phenotype, this strain is nonflagellated and non-motile. When compared to five known C. reinhardtii flagellar and basal body mutants, TLN1 was unique in having higher growth under nitrogen stress. The tln1 trait has potential benefits to large-scale algal and plant culturing under reduced nitrogen.

RpoS is a pleiotropic regulator of motility, biofilm formation, exoenzymes, siderophore and prodigiosin production, and trade-off during prolonged stationary phase in Serratia marcescens.

Prodigiosin is an important secondary metabolite produced by Serratia marcescens. It can help strains resist stresses from other microorganisms and environmental factors to achieve self-preservation. Prodigiosin is also a promising secondary metabolite due to its pharmacological characteristics. However, pigmentless S. marcescens mutants always emerge after prolonged starvation, which might be a way for the bacteria to adapt to starvation conditions, but it could be a major problem in the industrial application of S. marcescens. To identify the molecular mechanisms of loss of prodigiosin production, two mutants were isolated after 16 days of prolonged incubation of wild-type (WT) S. marcescens 1912768R; one mutant (named 1912768WR) exhibited reduced production of prodigiosin, and a second mutant (named 1912768W) was totally defective. Comparative genomic analysis revealed that the two mutants had either mutations or deletions in rpoS. Knockout of rpoS in S. marcescens 1912768R had pleiotropic effects. Complementation of rpoS in the ΔrpoS mutant further confirmed that RpoS was a positive regulator of prodigiosin production and that its regulatory role in prodigiosin biosynthesis was opposite that in Serratia sp. ATCC 39006, which had a different type of pig cluster; further, rpoS from Serratia sp. ATCC 39006 and other strains complemented the prodigiosin defect of the ΔrpoS mutant, suggesting that the pig promoters are more important than the genes in the regulation of prodigiosin production. Deletion of rpoS strongly impaired the resistance of S. marcescens to stresses but increased membrane permeability for nutritional competence; competition assays in rich and minimum media showed that the ΔrpoS mutant outcompeted its isogenic WT strain. All these data support the idea that RpoS is pleiotropic and that the loss of prodigiosin biosynthesis in S. marcescens 1912768R during prolonged incubation is due to a mutation in rpoS, which appears to be a self-preservation and nutritional competence (SPANC) trade-off.

Nonsynonymous Mutations in Linker-2 of the Pdr5 Multidrug Transporter Identify a New RNA Stability Element.

Analysis of synonymous mutations established that although the primary amino acid sequence remains unchanged, alterations in transcription and translation can result in significant phenotypic consequences. We report the novel observation that a series of nonsynonymous mutations in an unconserved stretch of amino acids found in the yeast multidrug efflux pump Pdr5 increases expression, thus enhancing multidrug resistance. Cycloheximide chase experiments ruled out the possibility that the increased steady-state level of Pdr5 was caused by increased protein stability. Quantitative-RT PCR experiments demonstrated that the mutants had levels of PDR5 transcript that were two to three times as high as in the isogenic wild-type strain. Further experiments employing metabolic labeling of mRNA with 4-thiouracil followed by uracil chasing showed that the half-life of PDR5 transcripts was specifically increased in these mutants. Our data demonstrate that the nucleotides encoding unconserved amino acids may be used to regulate expression and suggest that Pdr5 has a newly discovered RNA stability element within its coding region.

Effect of Nrg1 Repressor on NTH1 Transcription and Molecular Docking of Nrg1 on NTH1 Promoter

The amount of intracellular trehalose increases in response to environmental stress in yeast (Saccharomyces cerevisiae). When that stress is terminated, the accumulated trehalose rapidly degrades into glucose rapidly. Synthesis of trehalose is fulfilled by the Trehalose Phosphate Synthase (TPS) enzyme complex, whereas the degradation of trehalose is done by the neutral trehalase enzyme. Under different stress conditions, transcription of the NTH1 gene is activated and Stress Response Elements (STRE) are required for this activation. Nrg1 protein can bind promoters including STRE and PDS elements. Because of the presence of three possible Nrg1 repressor binding sites on the NTH1 promoter, the NTH1 gene may be regulated by the Nrg1 repressor. In order to test this hypothesis, Δnrg1 mutant yeast and its isogenic wild-type yeast strain were used to analyze the transcriptional activation of the NTH1 gene under nitrogen starving conditions. Nth1 transcription of the mutant yeast was seven-fold higher than that of the wild-type under growth conditions, and was not changed during nitrogen starvation. The protein-DNA docking analysis also supported the possibility of Nrg1 binding to the NTH1 promoter. These results revealed that NTH1 gene expression is constitutive in the absence of the Nrg1 repressor protein, hence the transcription of NTH1 is repressed by the Nrg1 protein.

An A666G mutation in transmembrane helix 5 of the yeast multidrug transporter Pdr5 increases drug efflux by enhancing cooperativity between transport sites.

Resistance to antimicrobial and chemotherapeutic agents is a significant clinical problem. Overexpression of multidrug efflux pumps often creates broad-spectrum resistance in cancers and pathogens. We describe a mutation, A666G, in the yeast ABC transporter Pdr5 that shows greater resistance to most of the tested compounds than does an isogenic wild-type strain. This mutant exhibited enhanced resistance without increasing either the amount of protein in the plasma membrane or the ATPase activity. In fluorescence quenching transport assays with rhodamine 6G in purified plasma membrane vesicles, the initial rates of rhodamine 6G fluorescence quenching of both the wild type and mutant showed a strong dependence on the ATP concentration, but were about twice as high in the latter. Plots of the initial rate of fluorescence quenching versus ATP concentration exhibited strong cooperativity that was further enhanced in the A666G mutant. Resistance to imazalil sulfate was about 3-4x as great in the A666G mutant strain as in the wild type. When this transport substrate was used to inhibit the rhodamine 6G transport, the A666G mutant inhibition curves also showed greater cooperativity than the wild-type strain. Our results suggest a novel and important mechanism: under selection, Pdr5 mutants can increase drug resistance by improving cooperative interactions between drug transport sites.

Small alarmones (p)ppGpp regulate virulence associated traits and pathogenesis of Salmonella enterica serovar Typhi.

How Salmonella enterica serovar Typhi (S.Typhi), an important human pathogen, survives the stressful microenvironments inside the gastrointestinal tract and within macrophages remains poorly understood. We report here that S.Typhi has a bonafide stringent response (SR) system, which is mediated by (p)ppGpp and regulates multiple virulence-associated traits and the pathogenicity of the S.Typhi Ty2 strain. In an iron overload mouse model of S.Typhi infection, the (p)ppGpp0 (Ty2ΔRelAΔSpoT) strain showed minimal systemic spread and no mortality, as opposed to 100% death of the mice challenged with the isogenic wild-type strain. Ty2ΔRelAΔSpoT had markedly elongated morphology with incomplete septa formation and demonstrated severely attenuated motility and chemotaxis due to the loss of flagella. Absence of the Vi-polysaccharide capsule rendered the mutant strain highly susceptible to complement-mediated lysis. The phenotypes of Ty2ΔRelAΔSpoT was contributed by transcriptional repression of several genes, including fliC, tviA, and ftsZ, as found by reverse transcriptase quantitative polymerase chain reaction and gene complementation studies. Finally, Ty2ΔRelAΔSpoT had markedly reduced invasion into intestinal epithelial cells and significantly attenuated survival within macrophages. To the best of our knowledge, this was the first study that addressed SR in S.Typhi and showed that (p)ppGpp was essential for optimal pathogenic fitness of the organism.

Do amyloid structures formed by Staphylococcus aureus phenol-soluble modulins have a biological function?

Phenol-soluble modulins (PSMs) are alpha-helical, amphipathic peptides that have multiple functions in staphylococcal physiology and virulence. Recent research has suggested that PSMs form amyloid fibrils and amyloids are involved in PSM-mediated phenotypes such as cytolysis and biofilm stability. While we observed PSM amyloid formation using electron microscopy and dye assays, there were no apparent differences in the production of extracellular fibrous material between a PSM-deficient strain and the isogenic wild-type strain. Furthermore, we detected no correlation between cytolytic or pro-inflammatory activities with the propensity of PSM derivatives to form amyloids. In addition, we propose a model based on our finding of non-specific attachment of PSMs to DNA, which we here report results in resistance to DNase digestion, explaining previous findings on PSM-mediated biofilm stability without the necessity to assume amyloid involvement. Collectively, our results indicate that PSM amyloid formation may not be of major relevance for known key biological functions of PSMs. Intriguingly, however, we found that amyloid-forming capacity of PSMalpha3 allows almost no amino acid exchanges, suggesting importance of amyloid formation in possibly yet unknown functions of PSMs.

RpoS role in virulence and fitness in enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) is a diarrheagenic pathogen that afflicts infants in developing countries. The most important virulence trait of EPEC is its ability to intimately adhere to cells in the small intestine, and to elicit diarrhea. The alternative sigma factor RpoS is involved in the virulence of several bacterial species. RpoS coordinates the general stress response and accumulates in cells under stress or in the stationary phase. RpoS levels differ across E. coli strains. High-RpoS strains are highly resistant to environmental stresses, but usually display low nutritional competence, while low-RpoS strains show the opposite phenotype. Here we investigated whether RpoS plays a role in the virulence and fitness of two different EPEC strains, E2348/69 and LRT9. A rpoS null mutation had a small positive effect on LRT9 adherence to epithelial cells, but the expression of the EPEC adhesins BfpA and intimin was not significantly affected by the mutation. E2348/69 adherence was not significantly affected by the rpoS mutation. The intrinsic level of RpoS was higher in LRT9 than in E2348/69 while the latter adhered more strongly and expressed higher levels of the adhesin BfpA than the former. Knockout of rpoS strongly impaired resistance to oxidative, osmotic and acid stress in both E2348/69 and LRT9. However, strain E2348/69 was significantly more sensitive to oxidative stress than LRT9. Finally, competition assays showed that the rpoS mutant of LRT9 displayed higher fitness under continuous culture than its isogenic wild-type strain, while E2348/69 outcompeted its rpoS mutant. In conclusion, RpoS plays mostly a positive role in EPEC biology and at least in the case of strain E2348/69 it is not constrained by the trade-off between vegetative growth and stress resistance.