Exposure of Legionella pneumophila to low-shear modeled microgravity: impact on stress response, membrane lipid composition, pathogenicity to macrophages and interrelated genes expression. - Related Documents




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80801.0000Exposure of Legionella pneumophila to low-shear modeled microgravity: impact on stress response, membrane lipid composition, pathogenicity to macrophages and interrelated genes expression. Here, we studied the effect of low-shear modeled microgravity (LSMMG) on cross stress resistance (heat, acid, and oxidative), fatty acid content, and pathogenicity along with alteration in expression of stress-/virulence-associated genes in Legionella pneumophila. The stress resistance analysis result indicated that bacteria cultivated under LSMMG environments showed higher resistance with elevated D-values at 55 °C and in 1 mM of hydrogen peroxide (H(2)O(2)) conditions compared to normal gravity (NG)-grown bacteria. On the other hand, there was no significant difference in tolerance (p < 0.05) toward simulated gastric fluid (pH-2.5) acid conditions. In fatty acid analysis, our result showed that a total amount of saturated and cyclic fatty acids was increased in LSMMG-grown cells; as a consequence, they might possess low membrane fluidity. An upregulated expression level was noticed for stress-related genes (hslV, htrA, grpE, groL, htpG, clpB, clpX, dnaJ, dnaK, rpoH, rpoE, rpoS, kaiB, kaiC, lpp1114, ahpC1, ahpC2, ahpD, grlA, and gst) under LSMMG conditions. The reduced virulence (less intracellular bacteria and less % of induce apoptosis in RAW 264.7 macrophages) of L. pneumophila under LSMMG conditions may be because of downregulation related genes (dotA, dotB, dotC, dotD, dotG, dotH, dotL, dotM, dotN, icmK, icmB, icmS, icmT, icmW, ladC, rtxA, letA, rpoN, fleQ, fleR, and fliA). In the LSMMG group, the expression of inflammation-related factors, such as IL-1α, TNF-α, IL-6, and IL-8, was observed to be reduced in infected macrophages. Also, scanning electron microscopy (SEM) analysis showed less number of LSMMG-cultivated bacteria attached to the host macrophages compared to NG. Thus, our study provides understandings about the changes in lipid composition and different genes expression due to LSMMG conditions, which apparently influence the alterations of L. pneumophila' stress/virulence response.202438305908
635210.9965Cloning and characterization of grpE in Acetobacter pasteurianus NBRC 3283. The grpE gene in Acetobacter pasteurianus NBRC 3283 was cloned and characterized, to elucidate the mechanism underlying the resistance of acetic acid bacteria to the stressors existing during acetic acid fermentation. This gene was found to be located in tandem with two related genes, appearing on the genome in the order grpE-dnaK-dnaJ. A sigma(32)-type promoter sequence was found in the upstream region of grpE. The relative transcription levels of grpE, dnaK, and dnaJ mRNA were in the ratio of approximately 1:2:0.1, and the genes were transcribed as grpE-dnaK, dnaK, and dnaJ. The transcription level of grpE was elevated by heat shock and treatment with ethanol. Co-overexpression of GrpE with DnaK/J in cells resulted in improved growth compared to the single overexpression of DnaK/J in high temperature or ethanol-containing conditions, suggesting that GrpE acts cooperatively with DnaK/J for expressing resistance to those stressors considered to exist during acetic acid fermentation. Our findings indicate that GrpE is closely associated with adaptation to stressors in A. pasteurianus and may play an important role in acetic acid fermentation.201020129077
66720.9959Increased intracellular H(2)S levels enhance iron uptake in Escherichia coli. We investigated the impact of intracellular hydrogen sulfide (H(2)S) hyperaccumulation on the transcriptome of Escherichia coli. The wild-type (WT) strain overexpressing mstA, encoding 3-mercaptopyruvate sulfur transferase, produced significantly higher H(2)S levels than the control WT strain. The mstA-overexpressing strain exhibited increased resistance to antibiotics, supporting the prior hypothesis that intracellular H(2)S contributes to oxidative stress responses and antibiotic resistance. RNA-seq analysis revealed that over 1,000 genes were significantly upregulated or downregulated upon mstA overexpression. The upregulated genes encompassed those associated with iron uptake, including siderophore synthesis and iron import transporters. The mstA-overexpressing strain showed increased levels of intracellular iron content, indicating that H(2)S hyperaccumulation affects iron availability within cells. We found that the H(2)S-/supersulfide-responsive transcription factor YgaV is required for the upregulated expression of iron uptake genes in the mstA-overexpression conditions. These findings indicate that the expression of iron uptake genes is regulated by intracellular H(2)S, which is crucial for oxidative stress responses and antibiotic resistance in E. coli. IMPORTANCE: H(2)S is recognized as a second messenger in bacteria, playing a vital role in diverse intracellular and extracellular activities, including oxidative stress responses and antibiotic resistance. Both H(2)S and iron serve as essential signaling molecules for gut bacteria. However, the intricate intracellular coordination between them, governing bacterial physiology, remains poorly understood. This study unveils a close relationship between intracellular H(2)S accumulation and iron uptake activity, a relationship critical for antibiotic resistance. We present additional evidence expanding the role of intracellular H(2)S synthesis in bacterial physiology.202439324809
67030.9958ZntR is a critical regulator for zinc homeostasis and involved in pathogenicity in Riemerella anatipestifer. Zinc (Zn(2+)) is essential for all bacteria, but excessive Zn(2+) levels are toxic. Bacteria maintain zinc homeostasis through regulators, such as Zur, AdcR, and ZntR. Riemerella anatipestifer is a significant Flavobacteriales pathogen causing acute serositis in ducks and other birds. In this study, we identified a homolog of ZntR, a regulator for zinc homeostasis, and demonstrated its contribution to the pathogenicity of R. anatipestifer. Deletion of zntR makes the bacteria hypersensitive to excess Zn(2+) but not to other metals like manganese (Mn(2+)), copper (Cu(2+)), cobalt (Co(2+)), and nickel (Ni(2+)). Deletion of zntR also leads to intracellular zinc accumulation but not of other metals. Additionally, compared to the wild type, the deletion of zntR increases resistance to oxidants hydrogen peroxide (H(2)O(2)) and sodium hypochlorite (NaOCl), respectively. The deletion of zntR causes significant changes in transcriptional and protein expression levels, revealing 35 genes with potential zinc metabolism functions. Among them, zupT, which is inhibited by ZntR, is required for zinc transport and resistance to oxidative stress. Finally, deletion of zntR leads to attenuation of colonization in ducklings. In summary, ZntR is a crucial regulator for zinc homeostasis and contributes to the pathogenicity of R. anatipestifer.IMPORTANCEZinc homeostasis plays a critical role in the environmental adaptability of bacteria. Riemerella anatipestifer is a significant pathogen in poultry with the potential to encounter zinc-deficient or zinc-excess environment. The mechanism of zinc homeostasis in this bacterium remains largely unexplored. In this study, we showed that the transcriptional regulator ZntR of R. anatipestifer is critical for zinc homeostasis by altering the transcription and expression of a number of genes. Importantly, ZntR inhibits the transcription of zinc transporter ZupT and contributes to colonization in R. anatipestifer. The results are significant for understanding zinc homeostasis and the pathogenic mechanisms in R. anatipestifer.202540035565
868040.9957Environmental pH affects transcriptional responses to cadmium toxicity in Escherichia coli K-12 (MG1655). It has been widely reported that pH mediates cadmium toxicity to bacteria. We used a tripartite approach to investigate mechanisms by which pH affects cadmium toxicity that included analyses of: (1) growth kinetics, (2) global gene expression, and (3) cadmium speciation. Cadmium extended the lag phase at pH 7, but not at pH 5. DNA microarray analysis revealed that stress response genes including hdeA, otsA, and yjbJ were more highly expressed at pH 5 than at pH 7 after only 5 min of exposure to cadmium, suggesting that acidic pH more rapidly induced genes that confer cadmium resistance. In addition, genes involved in transport and many hypothetical genes were more highly expressed at pH 5 than at pH 7 in the presence of cadmium. Concentrations of two cadmium species, including one previously implicated in the mechanism by which pH mediates cadmium toxicity (CdOH+), increased with pH. Our data demonstrate that transcriptional responses of Escherichia coli to cadmium are substantially affected by pH and suggest that several stress response, transport, and hypothetical genes play roles in the mechanism by which pH mediates cadmium toxicity.200919220470
353350.9957Effects of three strains of intestinal autochthonous bacteria and their extracellular products on the immune response and disease resistance of common carp, Cyprinus carpio. The study isolated three strains of intestinal autochthonous bacteria Aeromonas veronii BA-1, Vibrio lentus BA-2, and Flavobacterium sasangense BA-3 from the intestinal tract of the common carp (Cyprinus carpio). To reveal the effects of these three strains of bacteria on the innate immunity of carp, the lysozyme, complement C3, total serum protein, albumin and globulin levels, respiratory burst activity, phagocytic activity by blood leucocytes and the expression of IL-1b, lysozyme-C, and TNF-α were examined after feeding with seven different diets for up to 28 days. Also the survival of carp against Aeromonas hydrophila was challenged for 14 days. The carp were fed seven different diets: one control, three diets supplemented with 1 × 10(8) cell g(-1) of carp intestinal bacteria BA-1 (Group D-I), BA-2 (Group D-II) and BA-3 (Group D-III), and three diets supplemented with extracellular products FA-1 (Group E-I), FA-2 (Group E-II) and FA-3 (Group E-III) which were corresponding to the strains BA-1, BA-2, and BA-3, respectively, up to 28 days. For groups D-I, D-III, E-I and E-III, the innate immune parameters of carp were significantly increased, the expression of three immune-related genes in blood was significantly up-regulated examined during 7, 14, and 21 days of feeding, and the survival rate was improved. The study indicates that the two isolated intestinal autochthonous bacteria A. veronii BA-1 and F. sasangense BA-3 could positively influence immune response and enhance disease resistance of carp against A. hydrophila infection.201424161775
887760.9956Conditioning of uropathogenic Escherichia coli for enhanced colonization of host. While in transit within and between hosts, uropathogenic Escherichia coli (UPEC) encounters multiple stresses, including substantial levels of nitric oxide and reactive nitrogen intermediates. Here we show that UPEC, the primary cause of urinary tract infections, can be conditioned to grow at higher rates in the presence of acidified sodium nitrite (ASN), a model system used to generate nitrosative stress. When inoculated into the bladder of a mouse, ASN-conditioned UPEC bacteria are far more likely to establish an infection than nonconditioned bacteria. Microarray analysis of ASN-conditioned bacteria suggests that several NsrR-regulated genes and other stress- and polyamine-responsive factors may be partially responsible for this effect. Compared to K-12 reference strains, most UPEC isolates have increased resistance to ASN, and this resistance can be substantially enhanced by addition of the polyamine cadaverine. Nitrosative stress, as generated by ASN, can stimulate cadaverine synthesis by UPEC, and growth of UPEC in cadaverine-supplemented broth in the absence of ASN can also promote UPEC colonization of the bladder. These results suggest that UPEC interactions with polyamines or stresses such as reactive nitrogen intermediates can in effect reprogram the bacteria, enabling them to better colonize the host.200919255192
881970.9956Responses of Bacillus sp. under Cu(II) stress in relation to extracellular polymeric substances and functional gene expression level. The production and composition of extracellular polymeric substances (EPS), as well as the EPS-related functional resistance genes and metabolic levels of Bacillus sp. under Cu(II) stress, were investigated. EPS production increased by 2.73 ± 0.29 times compared to the control when the strain was treated with 30 mg L(-1) Cu(II). Specifically, the polysaccharide (PS) content in EPS increased by 2.26 ± 0.28 g CDW(-1) and the PN/PS (protein/polysaccharide) ratio value increased by 3.18 ± 0.33 times under 30 mg L(-1) Cu(II) compared to the control. The increased EPS secretion and higher PN/PS ratio in EPS strengthened the cells' ability to resist the toxic effect of Cu(II). Differential expression of functional genes under Cu(II) stress was revealed by Gene Ontology pathway enrichment analysis and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. The enriched genes were most obviously upregulated in the UMP biosynthesis pathway, the pyrimidine metabolism pathway, and the TCS metabolism pathway. This indicates an enhancement of EPS regulation-related metabolic levels and their role as a defense mechanism for cells to adapt to Cu(II) stress. Additionally, seven copper resistance genes were upregulated while three were downregulated. The upregulated genes were related to the heavy metal resistance, while downregulated genes were related to cell differentiation, indicating that the strain had initiated an obvious resistance to Cu(II) despite its severe cell toxicity. These results provided a basis for promoting EPS-regulated associated functional genes and the application of gene-regulated bacteria in heavy metal-containing wastewater treatment.202337195605
623080.9956dpr and sod in Streptococcus mutans are involved in coexistence with S. sanguinis, and PerR is associated with resistance to H2O2. Large numbers of bacteria coexist in the oral cavity. Streptococcus sanguinis, one of the major bacteria in dental plaque, produces hydrogen peroxide (H(2)O(2)), which interferes with the growth of other bacteria. Streptococcus mutans, a cariogenic bacterium, can coexist with S. sanguinis in dental plaque, but to do so, it needs a means of detoxifying the H(2)O(2) produced by S. sanguinis. In this study, we investigated the association of three oxidative stress factors, Dpr, superoxide dismutase (SOD), and AhpCF, with the resistance of S. sanguinis to H(2)O(2). The knockout of dpr and sod significantly increased susceptibility to H(2)O(2), while the knockout of ahpCF had no apparent effect on susceptibility. In particular, dpr inactivation resulted in hypersensitivity to H(2)O(2). Next, we sought to identify the factor(s) involved in the regulation of these oxidative stress genes and found that PerR negatively regulated dpr expression. The knockout of perR caused increased dpr expression levels, resulting in low-level susceptibility to H(2)O(2) compared with the wild type. Furthermore, we evaluated the roles of perR, dpr, and sod when S. mutans was cocultured with S. sanguinis. Culturing of the dpr or sod mutant with S. sanguinis showed a significant decrease in the S. mutans population ratio compared with the wild type, while the perR mutant increased the ratio. Our results suggest that dpr and sod in S. mutans are involved in coexistence with S. sanguinis, and PerR is associated with resistance to H(2)O(2) in regulating the expression of Dpr.201323263955
67990.9956RNA-Seq Analysis Discovers the Critical Role of Rel in ppGpp Synthesis, Pathogenicity, and the VBNC State of Clavibacter michiganensis. The viable but nonculturable (VBNC) state is a unique survival strategy of bacteria in response to stress conditions. It was confirmed that Clavibacter michiganensis, the causal agent of bacterial canker in tomato, could be induced into the VBNC state by exposure to CuSO(4) in an oligotrophic solution. RNA-sequencing analysis was used to monitor the mechanisms of the VBNC state during CuSO(4) induction in C. michiganensis. The results identified that numerous genes involved in stringent response, copper resistance, and stress resistance were upregulated, and some involved in cell division were downregulated significantly. The study investigated the importance of Rel, which is an essential enzyme in the synthesis of the molecular alarmone ppGpp, via the generation of a Δrel mutant and its complementation strain. Biological characterization revealed that deficiency of rel reduced the bacterial growth, production of exopolysaccharides, and pathogenicity as well as ppGpp production. The Δrel mutant increased the sensitivity to environmental stress, exhibiting reduced growth on minimal media and a propensity to enter the VBNC state in response to CuSO(4). These findings have important implications for the understanding of survival mechanism and management of C. michiganensis and other phytopathogenic bacteria.202235341314
613100.99564-Hydroxy-2-nonenal antimicrobial toxicity is neutralized by an intracellular pathogen. Pathogens encounter numerous antimicrobial responses during infection, including the reactive oxygen species (ROS) burst. ROS-mediated oxidation of host membrane poly-unsaturated fatty acids (PUFAs) generates the toxic alpha-beta carbonyl 4-hydroxy-2-nonenal (4-HNE). Although studied extensively in the context of sterile inflammation, research into 4-HNE's role during infection remains limited. Here, we found that 4-HNE is generated during bacterial infection, that it impacts growth and survival in a range of bacteria, and that the intracellular pathogen Listeria monocytogenes induces many genes in response to 4-HNE exposure. A component of the L. monocytogenes 4-HNE response is the expression of the genes lmo0103 and lmo0613, deemed rha1 and rha2 (reductase of host alkenals), respectively, which code for two NADPH-dependent oxidoreductases that convert 4-HNE to the product 4-hydroxynonanal (4-HNA). Loss of these genes had no impact on L. monocytogenes bacterial burdens during murine or tissue culture infection. However, heterologous expression of rha1/2 in Bacillus subtilis significantly increased bacterial resistance to 4-HNE in vitro and promoted bacterial survival following phagocytosis by murine macrophages in an ROS-dependent manner. Thus, Rha1 and Rha2 are not necessary for 4-HNE resistance in L. monocytogenes but are sufficient to confer resistance to an otherwise sensitive organism in vitro and in host cells. Our work demonstrates that 4-HNE is a previously unappreciated component of ROS-mediated toxicity encountered by bacteria within eukaryotic hosts.202133955352
693110.9955Effect of acid adaptation on the fate of Listeria monocytogenes in THP-1 human macrophages activated by gamma interferon. In Listeria monocytogenes the acid tolerance response (ATR) takes place through a programmed molecular response which ensures cell survival under unfavorable conditions. Much evidence links ATR with virulence, but the molecular determinants involved in the reactivity to low pHs and the behavior of acid-exposed bacteria within host cells are still poorly understood. We have investigated the effect of acid adaptation on the fate of L. monocytogenes in human macrophages. Expression of genes encoding determinants for cell invasion and intracellular survival was tested for acid-exposed bacteria, and invasive behavior in the human myelomonocytic cell line THP-1 activated with gamma interferon was assessed. Functional approaches demonstrated that preexposure to an acidic pH enhances the survival of L. monocytogenes in activated human macrophages and that this effect is associated with an altered pattern of expression of genes involved in acid resistance and cell invasion. Significantly decreased transcription of the plcA gene, encoding a phospholipase C involved in vacuolar escape and cell-to-cell spread, was observed in acid-adapted bacteria. This effect was due to a reduction in the quantity of the bicistronic plcA-prfA transcript, concomitant with an increase in the level(s) of the monocistronic prfA mRNA(s). The transcriptional shift from distal to proximal prfA promoters resulted in equal levels of the prfA transcript (and, as a consequence, of the inlA, hly, and actA transcripts) under neutral and acidic conditions. In contrast, the sodC and gad genes, encoding a cytoplasmic superoxide dismutase and the glutamate-based acid resistance system, respectively, were positively regulated at a low pH. Morphological approaches confirmed the increased intracellular survival and growth of acid-adapted L. monocytogenes cells both in vacuoles and in the cytoplasm of interferon gamma-activated THP-1 macrophages. Our data indicate that preexposure to a low pH has a positive impact on subsequent challenge of L. monocytogenes with macrophagic cells.200212117947
8817120.9955Study on the estradiol degradation gene expression and resistance mechanism of Rhodococcus R-001 under low-temperature stress. Estradiol (E2), an endocrine disruptor, acts by mimicking or interfering with the normal physiological functions of natural hormones within organisms, leading to issues such as endocrine system disruption. Notably, seasonal fluctuations in environmental temperature may influence the degradation speed of estradiol (E2) in the natural environment, intensifying its potential health and ecological risks. Therefore, this study aims to explore how bacteria can degrade E2 under low-temperature conditions, unveiling their resistance mechanisms, with the goal of developing new strategies to mitigate the threat of E2 to health and ecological safety. In this paper, we found that Rhodococcus equi DSSKP-R-001 (R-001) can efficiently degrade E2 at 30 °C and 10 °C. Six genes in R-001 were shown to be involved in E2 degradation by heterologous expression at 30 °C. Among them, 17β-HSD, KstD2, and KstD3, were also involved in E2 degradation at 10 °C; KstD was not previously known to degrade E2. RNA-seq was used to characterize differentially expressed genes (DEGs) to explore the stress response of R-001 to low-temperature environments to elucidate the strain's adaptation mechanism. At the low temperature, R-001 cells changed from a round spherical shape to a long rod or irregular shape with elevated unsaturated fatty acids and were consistent with the corresponding genetic changes. Many differentially expressed genes linked to the cold stress response were observed. R-001 was found to upregulate genes encoding cold shock proteins, fatty acid metabolism proteins, the ABC transport system, DNA damage repair, energy metabolism and transcriptional regulators. In this study, we demonstrated six E2 degradation genes in R-001 and found for the first time that E2 degradation genes have different expression characteristics at 30 °C and 10 °C. Linking R-001 to cold acclimation provides new insights and a mechanistic basis for the simultaneous degradation of E2 under cold stress in Rhodococcus adaptation.202438677604
682130.9955Comparative transcriptome analysis of Brucella melitensis in an acidic environment: Identification of the two-component response regulator involved in the acid resistance and virulence of Brucella. Brucella melitensis, encounters a very stressful environment in phagosomes, especially low pH levels. So identifying the genes that contribute to the replication and survival within an acidic environment is critical in understanding the pathogenesis of the Brucella bacteria. In our research, comparative transcriptome with RNA-seq were used to analyze the changes of genes in normal-medium culture and in pH4.4-medium culture. The results reveal that 113 genes expressed with significant differences (|log2Ratio| ≥ 3); about 44% genes expressed as up-regulated. With GO term analysis, structural constituent of the ribosome, rRNA binding, structural molecule activity, and cation-transporting ATPase activity were significantly enriched (p-value ≤ 0.05). These genes distributed in 51 pathways, in which ribosome and photosynthesis pathways were significantly enriched. Six pathways (oxidative phosphorylation, iron-transporting, bacterial secretion system, transcriptional regulation, two-component system, and ABC transporters pathways) tightly related to the intracellular survival and virulence of Brucella were analyzed. A two-component response regulator gene in the transcriptional regulation pathway, identified through gene deletion and complementary technologies, played an important role in the resistance to the acid-resistance and virulence of Brucella.201626691825
692140.9955The ArcA regulon and oxidative stress resistance in Haemophilus influenzae. Haemophilus influenzae transits between niches within the human host that are predicted to differ in oxygen levels. The ArcAB two-component signal transduction system controls gene expression in response to respiratory conditions of growth and has been implicated in bacterial pathogenesis, yet the mechanism is not understood. We undertook a genome-scale study to identify genes of the H. influenzae ArcA regulon. Deletion of arcA resulted in increased anaerobic expression of genes of the respiratory chain and of H. influenzae's partial tricarboxylic acid cycle, and decreased anaerobic expression levels of genes of polyamine metabolism, and iron sequestration. Deletion of arcA also conferred a susceptibility to transient exposure to hydrogen peroxide that was greater following anaerobic growth than after aerobic growth. Array data revealed that the dps gene, not previously assigned to the ArcA modulon in bacteria, exhibited decreased expression in the arcA mutant. Deletion of dps resulted in hydrogen peroxide sensitivity and complementation restored resistance, providing insight into the previously uncharacterized mechanism of arcA-mediated H(2)O(2) resistance. The results indicate a role for H. influenzae arcA and dps in pre-emptive defence against transitions from growth in low oxygen environments to aerobic exposure to hydrogen peroxide, an antibacterial oxidant produced by phagocytes during infection.200717542927
669150.9955Manganese Efflux Achieved by MetA and MetB Affects Oxidative Stress Resistance and Iron Homeostasis in Riemerella anatipestifer. In bacteria, manganese homeostasis is controlled by import, regulation, and efflux. Here, we identified 2 Mn exporters, MetA and MetB (manganese efflux transporters A and B), in Riemerella anatipestifer CH-1, encoding a putative cation diffusion facilitator (CDF) protein and putative resistance-nodulation-division (RND) efflux pump, respectively. Compared with the wild type (WT), ΔmetA, ΔmetB, and ΔmetAΔmetB exhibited sensitivity to manganese, since they accumulated more intracellular Mn(2+) than the WT under excess manganese conditions, while the amount of iron in the mutants was decreased. Moreover, ΔmetA, ΔmetB, and ΔmetAΔmetB were more sensitive to the oxidant NaOCl than the WT. Further study showed that supplementation with iron sources could alleviate manganese toxicity and that excess manganese inhibited bacterial cell division. RNA-Seq showed that manganese stress resulted in the perturbation of iron metabolism genes, further demonstrating that manganese efflux is critical for iron homeostasis. metA transcription was upregulated under excess manganese but was not activated by MetR, a DtxR family protein, although MetR was also involved in manganese detoxification, while metB transcription was downregulated under iron depletion conditions and in fur mutants. Finally, homologues of MetA and MetB were found to be mainly distributed in members of Flavobacteriaceae. Specifically, MetB represents a novel manganese exporter in Gram-negative bacteria. IMPORTANCE Manganese is required for the function of many proteins in bacteria, but in excess, manganese can mediate toxicity. Therefore, the intracellular levels of manganese must be tightly controlled. Manganese efflux transporters have been characterized in some other bacteria; however, their homologues could not be found in the genome of Riemerella anatipestifer through sequence comparison. This indicated that other types of manganese efflux transporters likely exist. In this study, we characterized 2 transporters, MetA and MetB, that mediate manganese efflux in R. anatipestifer in response to manganese overload. MetA encodes a putative cation diffusion facilitator (CDF) protein, which has been characterized as a manganese transporter in other bacteria, while this is the first observation of a putative resistance-nodulation-division (RND) transporter contributing to manganese export in Gram-negative bacteria. In addition, the mechanism of manganese toxicity was studied by observing morphological changes and by transcriptome sequencing. Taken together, these results are important for expanding our understanding of manganese transporters and revealing the mechanism of manganese toxicity.202336815770
6233160.9955Exploring the response of Escherichia coli O157:H7 EDL933 within Acanthamoeba castellanii by genome-wide transcriptional profiling. Free-living protozoa, such as Acanthamoeba castellanii, are environmental hosts for pathogenic bacteria. Protozoa have been implicated in harboring pathogenic bacteria and enhancing virulence factors and antibiotic resistance. To better understand this relationship with Escherichia coli O157:H7, we characterized its transcriptome within A. castellanii compared with broth-grown organisms using two-color microarrays. Statistical analysis indicated that 969 genes were differentially expressed at P<0.018, with a false discovery rate of 1.9% and a fold change cutoff of 1.3 or greater. There were 655 upregulated transcripts that include 40 genes associated with virulence, of which 32 are encoded on O-islands, and include shiga toxin genes (stx1A, stx1B stx2A) and 14 genes involved in Type III secretion system components. Also included are SOS response genes such as lexA and recA, genes involved in or predicted to be involved in antibiotic resistance (rarD, macAB, marABR, mdtK, yojI, yhgN), the quorum-sensing operon lsrACDB, and the efe and feo iron-acquisition systems. There were 314 downregulated transcripts that included 19 transcripts associated with virulence, seven of which are encoded on O-islands. Our results demonstrate that a significant portion of the E. coli O157:H7 genome was differentially expressed as a result of the protozoan intracellular environment.201020831595
668170.9954c-di-GMP regulates the resistance of Pseudomonas aeruginosa to heat shock and aminoglycoside antibiotics by targeting the σ factor RpoH. Cyclic di-GMP (c-di-GMP) is a second messenger molecule that is widely distributed in bacteria and plays various physiologically important regulatory roles through interactions with a variety of effector molecules. Sigma (σ) factors are the predominant transcription factors involved in transcription regulation in bacteria. While c-di-GMP has been shown to bind to a range of transcription factors, c-di-GMP-binding σ factors have never been reported before. In a c-di-GMP/σ factors binding screen, we identified the σ factor RpoH as a c-di-GMP-responsive transcription factor in Pseudomonas aeruginosa PAO1. We further show that the binding of c-di-GMP to RpoH inhibits binding of RpoH to the promoters of its target genes such as asrA and dnaK, thereby downregulating the expression of these genes and reducing the resistance of P. aeruginosa to heat shock and aminoglycoside antibiotics. RpoH from Escherichia coli, Burkholderia thailandensis and Agrobacterium tumefaciens are also capable of binding c-di-GMP, suggesting that c-di-GMP-mediated control of the activity of RpoH is conserved in members of Proteobacteria.202641005124
6224180.9954Bacteriophage-resistant Staphylococcus aureus mutant confers broad immunity against staphylococcal infection in mice. In the presence of a bacteriophage (a bacteria-attacking virus) resistance is clearly beneficial to the bacteria. As expected in such conditions, resistant bacteria emerge rapidly. However, in the absence of the phage, resistant bacteria often display reduced fitness, compared to their sensitive counterparts. The present study explored the fitness cost associated with phage-resistance as an opportunity to isolate an attenuated strain of S. aureus. The phage-resistant strain A172 was isolated from the phage-sensitive strain A170 in the presence of the M(Sa) phage. Acquisition of phage-resistance altered several properties of A172, causing reduced growth rate, under-expression of numerous genes and production of capsular polysaccharide. In vivo, A172 modulated the transcription of the TNF-alpha, IFN-gamma and Il-1beta genes and, given intramuscularly, protected mice from a lethal dose of A170 (18/20). The heat-killed vaccine also afforded protection from heterologous methicillin-resistant S. aureus (MRSA) (8/10 mice) or vancomycin-intermediate S. aureus (VISA) (9/10 mice). The same vaccine was also effective when administered as an aerosol. Anti-A172 mouse antibodies, in the dose of 10 microl/mouse, protected the animals (10/10, in two independent experiments) from a lethal dose of A170. Consisting predominantly of the sugars glucose and galactose, the capsular polysaccharide of A172, given in the dose of 25 microg/mouse, also protected the mice (20/20) from a lethal dose of A170. The above results demonstrate that selection for phage-resistance can facilitate bacterial vaccine preparation.201020661301
6226190.9954Chlorhexidine Promotes Psl Expression in Pseudomonas aeruginosa That Enhances Cell Aggregation with Preserved Pathogenicity Demonstrates an Adaptation against Antiseptic. Because Pseudomonas aeruginosa is frequently in contact with Chlorhexidine (a regular antiseptic), bacterial adaptations are possible. In comparison with the parent strain, the Chlorhexidine-adapted strain formed smaller colonies with metabolic downregulation (proteomic analysis) with the cross-resistance against colistin (an antibiotic for several antibiotic-resistant bacteria), partly through the modification of L-Ara4N in the lipopolysaccharide at the outer membrane. Chlorhexidine-adapted strain formed dense liquid-solid interface biofilms with enhanced cell aggregation partly due to the Chlorhexidine-induced overexpression of psl (exopolysaccharide-encoded gene) through the LadS/GacSA pathway (c-di-GMP-independence) in 12 h biofilms and maintained the aggregation with SiaD-mediated c-di-GMP dependence in 24 h biofilms as evaluated by polymerase chain reaction (PCR). The addition of Ca(2+) in the Chlorhexidine-adapted strain facilitated several Psl-associated genes, indicating an impact of Ca(2+) in Psl production. The activation by Chlorhexidine-treated sessile bacteria demonstrated a lower expression of IL-6 and IL-8 on fibroblasts and macrophages than the activation by the parent strain, indicating the less inflammatory reactions from Chlorhexidine-exposed bacteria. However, the 14-day severity of the wounds in mouse caused by Chlorhexidine-treated bacteria versus the parent strain was similar, as indicated by wound diameters and bacterial burdens. In conclusion, Chlorhexidine induced psl over-expression and colistin cross-resistance that might be clinically important.202235955437