Pulse Generation in the Quorum Machinery of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Pseudomonas aeruginosa is a Gram-negative bacterium that is responsible for a wide range of infections in humans. Colonies employ quorum sensing (QS) to coordinate gene expression, including for virulence factors, swarming motility and complex social traits. The QS signalling system of P. aeruginosa is known to involve multiple control components, notably the las, rhl and pqs systems. In this paper, we examine the las system and, in particular, the repressive interaction of rsaL, an embedded small regulative protein, employing recent biochemical information to aid model construction. Using analytic methods, we show how this feature can give rise to excitable pulse generation in this subsystem with important downstream consequences for rhamnolipid production. We adopt a symmetric competitive inhibition to capture the binding in the lasI–rsaL intergenic region and show our results are not dependent on the exact choice of this functional form. Furthermore, we examine the coupling of lasR to the rhl system, the impact of the predicted capacity for pulse generation and the biophysical consequences of this behaviour. We hypothesize that the interaction between the las and rhl systems may provide a quorum memory to enable cells to trigger rhamnolipid production only when they are at the edge of an established aggregation.     

Equisetin as potential quorum sensing inhibitor of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Objective

To screen for the quorum-sensing (QS) inhibitors from marine-derived fungi and evaluate their anti-QS properties in Pseudomonas aeruginosa.

Results

QS inhibitory activity was found in secondary metabolites of a marine fungus Fusarium sp. Z10 using P. aeruginosa QSIS-lasI biosensor. The major active compound of this fungus was isolated by HPLC and identified as equisetin. Subinhibitory concentration of equisetin could inhibit the formation of biofilm, swarming motility, and the production of virulence factors in P. aeruginosa. The inhibition of las, PQS, and rhl system by equisetin were determined using Escherichia coli MG4/pKDT17, E.coli pEAL08-2, and E.coli pDSY, respectively. Real–time RT-PCR assays showed that equisetin could downregulate the mRNA expression of QS-related genes.

Conclusions

Equisetin proved its potential as an inhibitor against P. aeruginosa QS system and might also serve as precursor compound in development of novel therapeutics for infectious diseases by optimal design of structures.

     

Leaf Extracts of Selected Gardening Trees Can Attenuate Quorum Sensing and Pathogenicity of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PAO1

An increasing concern on resistance to multiple-antibiotics has led to the discovery of novel agents and the establishment of new precaution strategy. Numerous plant sources have been widely studied to reduce virulence of pathogenic bacteria by interfering cell-to-cell based communication called quorum sensing (QS). Leaf extracts of 17 gardening trees were collected and investigated for their anti-QS effects using a sensor strain Chromobacterium violaceum CV026. Methanolic extracts of K4 (Acer palmatum), K9 (Acer pseudosieboldianum) and K13 (Cercis chinensis) leaves were selected for further experiments based on their antagonism effect on QS without inhibiting C. violaceum CV026 growth. Subsequently, the leaf extracts on QS-mediated virulence of Pseudomonas aeruginosa PAO1 involved in biofilm formation, motility, bioluminescence, pyocyanin production, QS molecules production, and Caenorhabditis elegans killing activity were evaluated. The biofilm formation ability and swarming motility of P. aeruginosa PAO1 were decreased approximately 50% in the presence of these leaf extracts at a concentration of 1 mg/mL. The expression level of lecA::lux of P. aeruginosa PAO1 and pyocyanin production were also reduced. The three leaf extracts also decreased autoinducer (AI) production in P. aeruginosa PAO1 without direct degradation, suggesting that AI synthesis might have been suppressed by these extracts. The three leaf extracts also showed anti-infection activity in C. elegans model. Taken together, these results suggest that methanolic leaf extracts of K4, K9 and K13 have the potential to attenuate the virulence of P. aeruginosa PAO1.     

<Emphasis Type="Italic">Drosophila melanogaster</Emphasis> as a polymicrobial infection model for <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> and <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Non-mammalian infection models have been developed over the last two decades, which is a historic milestone to understand the molecular basis of bacterial pathogenesis. They also provide small-scale research platforms for identification of virulence factors, screening for antibacterial hits, and evaluation of antibacterial efficacy. The fruit fly, Drosophila melanogaster is one of the model hosts for a variety of bacterial pathogens, in that the innate immunity pathways and tissue physiology are highly similar to those in mammals. We here present a relatively simple protocol to assess the key aspects of the polymicrobial interaction in vivo between the human opportunistic pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, which is based on the systemic infection by needle pricking at the dorsal thorax of the flies. After infection, fly survival and bacteremia over time for both P. aeruginosa and S. aureus within the infected flies can be monitored as a measure of polymicrobial virulence potential. The infection takes ~24 h including bacterial cultivation. Fly survival and bacteremia are assessed using the infected flies that are monitored up to ~60 h post-infection. These methods can be used to identify presumable as well as unexpected phenotypes during polymicrobial interaction between P. aeruginosa and S. aureus mutants, regarding bacterial pathogenesis and host immunity.     

A common evolutionary pathway for maintaining quorum sensing in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

In the bacterium Pseudomonas aeruginosa, the synthesis and secretion of extracellular protease is a typical cooperative behavior regulated by quorum sensing. However, this type of cooperative behavior is easily exploited by other individuals who do not synthesize public goods, which is known as the “tragedy of the commons”. Here P. aeruginosa was inoculated into casein media with different nitrogen salts added. In casein broth, protease (a type of public good) is necessary for bacterial growth. After 30 days of sequential transfer, some groups propagated stably and avoided “tragedy of the commons”. The evolved cooperators who continued to synthesize protease were isolated from these stable groups. By comparing the characteristics of quorum sensing in these cooperators, an identical evolutionary pattern was found. A variety of cooperative behaviors regulated by quorum sensing, such as the synthesis and secretion of protease and signals, were significantly reduced during the process of evolution. Such reductions improved the efficiency of cooperation, helping to prevent cheating. In addition, the production of pyocyanin, which is regulated by the RhlIR system, increased during the process of evolution, possibly due to its role in stabilizing the cooperation. This study contributes towards our understanding of the evolution of quorum sensing of P. aeruginosa.     

Detection of <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> virulence genes in <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> subsp. <Emphasis Type="Italic">equisimilis</Emphasis> from Vellore,India

Streptococcus dysgalactiae subsp. equisimilis (SDSE), belonging to the group C and G streptococci, are human pathogens reported to cause clinical manifestations similar to infections caused by Streptococcus pyogenes. To scrutinize the distribution of gene coding for S. pyogenes virulence factors in SDSE, 255 isolates were collected from humans infected with SDSE in Vellore, a region in southern India, with high incidence of SDSE infections. Initial evaluation indicated SDSE isolates comprising of 82.35% group G and 17.64% group C. A multiplex PCR system was used to detect 21 gene encoding virulence-associated factors of S. pyogenes, like superantigens, DNases, proteinases, and other immune modulatory toxins. As validated by DNA sequencing of the PCR products, sequences homologous to speC, speG, speH, speI, speL, ssa and smeZ of the family of superantigen coding genes and for DNases like sdaD and sdc were detected in the SDSE collection. Furthermore, there was high abundance (48.12% in group G and 86.6% in group C SDSE) of scpA, the gene coding for C5a peptidase in these isolates. Higher abundance of S. pyogenes virulence factor genes was observed in SDSE of Lancefield group C as compared to group G, even though the incidence rates in former were lower. This study not only substantiates detection of S. pyogenes virulence factor genes in whole genome sequenced SDSE but also makes significant contribution towards the understanding of SDSE and its increasing virulence potential.     

Association between <Emphasis Type="Italic">cagA</Emphasis>, <Emphasis Type="Italic">vacAi</Emphasis>, and <Emphasis Type="Italic">dupA</Emphasis> genes of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> and gastroduodenal pathologies in Chilean patients

In addition to the already known cagA gene, novel genetic markers have been associated with Helicobacter pylori (H. pylori) virulence: the dupA and vacAi genes. These genes might play an important role as specific markers to determine the clinical outcome of the disease, especially the vacAi gene, which has been expected to be a good marker of severe pathologies like gastric adenocarcinoma. In the present study, the association of cagA, dupA, and vacAi genes with gastroduodenal pathologies in Chilean patients was studied. One hundred and thirty-two patients positive for H. pylori were divided into two groups—non-severe and severe gastric pathologies—and investigated for the presence of cagA, dupA, and vacAi H. pylori virulence genes by PCR. The cagA gene was detected in 20/132 patients (15.2%), the vacAi1 gene was detected in 54/132 patients (40.9%), the vacAi2 gene was detected in 26/132 patients (19.7%), and the dupA gene was detected in 50/132 (37.9%) patients. Logistic regression model analysis showed that the vacAi1 isoform gene in the infected strains and the severity of the diseases outcome were highly associated, causing severe gastric damage that may lead to gastric cancer (p < 0.0001; OR = 8.75; 95% CI 3.54–21.64). Conversely, cagA (p = 0.3507; OR = 1.62; 95% CI 0.59–4.45) and vacAi2 (p = 0.0114; OR = 3.09; 95% CI 1.26–7.60) genes were not associated with damage, while the dupA gene was associated significantly with non-severe clinical outcome (p = 0.0032; OR = 0.25; 95% CI 0.09–0.65). In addition, dupA gene exerts protection against severe gastric pathologies induced by vacAi1 by delaying the outcome of the disease by approximately 20 years.     

Usefulness of the Non-conventional <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> Model to Assess <Emphasis Type="Italic">Candida</Emphasis> Virulence

Invasive candidiasis is caused mainly by Candida albicans, but other Candida species have increasing etiologies. These species show different virulence and susceptibility levels to antifungal drugs. The aims of this study were to evaluate the usefulness of the non-conventional model Caenorhabditis elegans to assess the in vivo virulence of seven different Candida species and to compare the virulence in vivo with the in vitro production of proteinases and phospholipases, hemolytic activity and biofilm development capacity. One culture collection strain of each of seven Candida species (C. albicans, Candida dubliniensis, Candida glabrata, Candida krusei, Candida metapsilosis, Candida orthopsilosis and Candida parapsilosis) was studied. A double mutant C. elegans AU37 strain (glp-4;sek-1) was infected with Candida by ingestion, and the analysis of nematode survival was performed in liquid medium every 24 h until 120 h. Candida establishes a persistent lethal infection in the C. elegans intestinal tract. C. albicans and C. krusei were the most pathogenic species, whereas C. dubliniensis infection showed the lowest mortality. C. albicans was the only species with phospholipase activity, was the greatest producer of aspartyl proteinase and had a higher hemolytic activity. C. albicans and C. krusei caused higher mortality than the rest of the Candida species studied in the C. elegans model of candidiasis.     

<Emphasis Type="Italic">Aeromonas</Emphasis> and <Emphasis Type="Italic">Plesiomonas</Emphasis> species from scarlet ibis (<Emphasis Type="Italic">Eudocimus ruber</Emphasis>) and their environment: monitoring antimicrobial susceptibility and virulence

The present study aimed at evaluating the role of captive scarlet ibises (Eudocimus ruber) and their environment as reservoirs of Aeromonas spp. and Plesiomonas spp., and analyzing the in vitro antimicrobial susceptibility and virulence of the recovered bacterial isolates. Thus, non-lactose and weak-lactose fermenting, oxidase positive Gram-negative bacilli were recovered from cloacal samples (n = 30) of scarlet ibises kept in a conservational facility and from water samples (n = 30) from their environment. Then, the antimicrobial susceptibility, hemolytic activity and biofilm production of the recovered Aeromonas spp. and Plesiomonas shigelloides strains were assessed. In addition, the virulence-associated genes of Aeromonas spp. were detected. Ten Aeromonas veronii bv. sobria, 2 Aeromonas hydrophila complex and 10 P. shigelloides were recovered. Intermediate susceptibility to piperacillin-tazobactam and cefepime was observed in 2 Aeromonas spp. and 1 P. shigelloides, respectively, and resistance to gentamicin was observed in 4 P. shigelloides. The automated susceptibility analysis revealed resistance to piperacillin-tazobactam and meropenem among Aeromonas spp. and intermediate susceptibility to gentamicin among P. shigelloides. All Aeromonas isolates presented hemolytic activity, while 3 P. shigelloides were non-hemolytic. All Aeromonas spp. and 3/10 P. shigelloides were biofilm-producers, at 28 °C, while 10 Aeromonas spp. and 6/10 P. shigelloides produced biofilms, at 37 °C. The most prevalent virulence genes of Aeromonas spp. were asa1 and ascV. Scarlet ibises and their environment harbour potentially pathogenic bacteria, thus requiring monitoring and measures to prevent contamination of humans and other animals.     

Reactive oxygen species (ROS) and antioxidative enzyme status in <Emphasis Type="Italic">Solanum lycopersicum</Emphasis> on priming with fluorescent <Emphasis Type="Italic">Pseudomonas</Emphasis> spp. against <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

In plants, ROS signaling and increase in activities of antioxidants are among defense responses. The present study describes the oxidative stress profiling in model host plant tomato (Solanum lycopersicum L.), during an invasion of the wilt pathogen Fusarium oxysporum f. sp. lycopersici with or without seed priming with Pseudomonas isolates M80, M96 and T109. Tomato seeds were primed with known Pseudomonas isolates M80, M96 and T109 and the forty-day- old plants were challenged with spores of F. oxysporum under greenhouse conditions. Leaf samples were collected at 0, 24, 48 72 and 96 h post fungal challenge and analysed for systemic level of oxidative stress parameters including total phenolics, proline, hydrogen peroxide, lipid peroxidation and enzymatic antioxidants. Disease incidence in the plants under greenhouse conditions was also calculated. Results revealed that priming with Pseudomonas isolates resulted in reduced oxidative stress in the host, during pathogen invasion. M80-priming showed highest antioxidative protection to the host plants during F. oxysporum invasion. The observed reduction in hydrogen peroxide and lipid peroxidation in primed plants was in agreement with the increased activities of the corresponding antioxidant enzymes. Greenhouse results showed that the highest wilt disease symptoms were with M80-priming followed by M96 and T109. The present study gives substantial evidences on the oxidative stress mitigation in response to Pseudomonas-priming on the model tomato-Fusarium interaction system.     

Transferable Carbenicillin Resistance in <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

IN 1969, after carbenicillin had been in use for three years in this unit, highly resistant strains of Pseudomonas aeruginosa were isolated for the first time¹. Because these resistant strains included, from their first appearance, representatives of two unrelated types, it seemed likely that the resistance was transferable; this hypothesis was supported by experiments showing the transfer of carbenicillin resistance between Ps. aeruginosa and Escherichia coli K12 in vitro and in vivo^2–4;. The resistant Ps. aeruginosa produced a penicillinase (β lactamase) similar to that normally produced by some strains of Enterobacteria and different from that normally produced by Ps. aeruginosa^2,3, so it seemed likely that the Ps. aeruginosa had initially acquired resistance by the transfer of an R factor from a carbenicillin-resistant member of the Enterobacteriaceae colonizing the same burn. This hypothesis is now supported by a study on strains of Enterobacteria and Ps. aeruginosa isolated in a number of hospitals. We have also found evidence suggesting that Ps. aeruginosa which has acquired this R factor may not show resistance until it has been exposed repeatedly to carbenicillin.     

Clinical strains of <Emphasis Type="Italic">Lactobacillus</Emphasis> reduce the filamentation of <Emphasis Type="Italic">Candida albicans</Emphasis> and protect <Emphasis Type="Italic">Galleria mellonella</Emphasis> against experimental candidiasis

Candida albicans is the most common human fungal pathogen and can grow as yeast or filaments, depending on the environmental conditions. The filamentous form is of particular interest because it can play a direct role in adherence and pathogenicity. Therefore, the purpose of this study was to evaluate the effects of three clinical strains of Lactobacillus on C. albicans filamentation as well as their probiotic potential in pathogen-host interactions via an experimental candidiasis model study in Galleria mellonella. We used the reference strain Candida albicans ATCC 18804 and three clinical strains of Lactobacillus: L. rhamnosus strain 5.2, L. paracasei strain 20.3, and L. fermentum strain 20.4. First, the capacity of C. albicans to form hyphae was tested in vitro through association with the Lactobacillus strains. After that, we verified the ability of these strains to attenuate experimental candidiasis in a Galleria mellonella model through a survival curve assay. Regarding the filamentation assay, a significant reduction in hyphae formation of up to 57% was observed when C. albicans was incubated in the presence of the Lactobacillus strains, compared to a control group composed of only C. albicans. In addition, when the larvae were pretreated with Lactobacillus spp. prior to C. albicans infection, the survival rate of G. mellonela increased in all experimental groups. We concluded that Lactobacillus influences the growth and expression C. albicans virulence factors, which may interfere with the pathogenicity of these microorganisms.     

Contribution of the non-effector members of the HrpL regulon, <Emphasis Type="Italic">iaaL</Emphasis> and <Emphasis Type="Italic">matE</Emphasis>, to the virulence of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. tomato DC3000 in tomato plants

Background

The phytohormone indole-3-acetic acid (IAA) is widely distributed among plant-associated bacteria. Certain strains of the Pseudomonas syringae complex can further metabolize IAA into a less biologically active amino acid conjugate, 3-indole-acetyl-ε-L-lysine, through the action of the iaaL gene. In P. syringae and Pseudomonas savastanoi strains, the iaaL gene is found in synteny with an upstream gene, here called matE, encoding a putative MATE family transporter. In P. syringae pv. tomato (Pto) DC3000, a pathogen of tomato and Arabidopsis plants, the HrpL sigma factor controls the expression of a suite of virulence-associated genes via binding to hrp box promoters, including that of the iaaL gene. However, the significance of HrpL activation of the iaaL gene in the virulence of Pto DC3000 is still unclear.

Results

A conserved hrp box motif is found upstream of the iaaL gene in the genomes of P. syringae strains. However, although the promoter region of matE is only conserved in genomospecies 3 of this bacterial group, we showed that this gene also belongs to the Pto DC3000 HrpL regulon. We also demonstrated that the iaaL gene is transcribed both independently and as part of an operon with matE in this pathogen. Deletion of either the iaaL or the matE gene resulted in reduced fitness and virulence of Pto DC3000 in tomato plants. In addition, we used multicolor fluorescence imaging to visualize the responses of tomato plants to wild-type Pto DC3000 and to its ΔmatE and ΔiaaL mutants. Activation of secondary metabolism prior to the development of visual symptoms was observed in tomato leaves after bacterial challenges with all strains. However, the observed changes were strongest in plants challenged by the wild-type strain, indicating lower activation of secondary metabolism in plants infected with the ΔmatE or ΔiaaL mutants.

Conclusions

Our results provide new evidence for the roles of non-type III effector genes belonging to the Pto DC3000 HrpL regulon in virulence.

     

<Emphasis Type="Italic">In silico</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> analysis reveal a novel gene in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> trehalose metabolism

Background

The ability to respond rapidly to fluctuations in environmental changes is decisive for cell survival. Under these conditions trehalose has an essential protective function and its concentration increases in response to enhanced expression of trehalose synthase genes, TPS1, TPS2, TPS3 and TSL1. Intriguingly, the NTH1 gene, which encodes neutral trehalase, is highly expressed at the same time. We have previously shown that trehalase remains in its inactive non-phosphorylated form by the action of an endogenous inhibitor. Recently, a comprehensive two-hybrid analysis revealed a 41-kDa protein encoded by the YLR270w ORF, which interacts with NTH1p.

Results

In this work we investigate the correlation of this Trehalase Associated Protein, in trehalase activity regulation. The neutral trehalase activity in the ylr270w mutant strain was about 4-fold higher than in the control strain. After in vitro activation by PKA the ylr270w mutant total trehalase activity increased 3-fold when compared to a control strain. The expression of the NTH1 gene promoter fused to the heterologous reporter lacZ gene was evaluated. The mutant strain lacking YLR270w exhibited a 2-fold increase in the NTH1-lacZ basal expression when compared to the wild type strain.

Conclusions

These results strongly indicate a central role for Ylr270p in inhibiting trehalase activity, as well as in the regulation of its expression preventing a wasteful futile cycle of synthesis-degradation of trehalose.

     

Identification and genomic analysis of antifungal property of a tomato root endophyte <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. p21

Pseudomonas sp., which occupy a variety of ecological niches, have been widely studied for their versatile metabolic capacity to promote plant growth, suppress microbial pathogens, and induce systemic resistance in plants. In this study, a Pseudomonas sp. strain p21, which was isolated from tomato root endophytes, was identified as having antagonism against Aspergillus niger. Further analysis showed that this strain had the ability to biosynthesise siderophores and was less effective in inhibiting the growth of A. niger with the supplementation of Fe³⁺ in the agar medium. Genomic sequencing and the secondary metabolite cluster analysis demonstrated that Pseudomonas sp. p21 harboured 2 pyoverdine biosynthetic gene clusters, which encode compounds with predicted core structures and two variable tetra-peptide or eleven-peptide chains. The results indicated that siderophore-mediated competition for iron might be an important mechanism in Pseudomonas suppression of the fungal pathogen A. niger and in microbe-pathogen-plant interactions.