Yersinia Pestis Response to Macrophage-induced Stress from Hosts with High and Low Susceptibility to Plague

Yersinia pestis causes severe disease in natural rodent hosts but mild to inapparent disease in rodent predators such as dogs. Y. pestis initiates infection in susceptible hosts by parasitizing and multiplying intracellularly in local macrophages during the early stage of infection. Thus, we hypothesized that Y. pestis infection severity may depend on how well the intracellular bacterium overcomes the initial host macrophage associated stress. To test this hypothesis, Y. pestis infection progress was studied in mouse splenic and dog peripheral blood macrophages, and various parameters of this infection were measured in mouse and dog tissue culture macrophages RAW264.7 and DH82, respectively. The study showed that during the early stage of infection, intracellular Y. pestis assumed filamentous cellular morphology with multiple genomes per bacterium in both mouse and dog macrophages. Later, in mouse macrophages, these filamentous Y. pestis returned to coccobacilli with spacious vacuolar extension ofVeterinary Pathobiolog

Understanding the molecular basis of plant growth promotional effect of Pseudomonas fluorescens on rice through protein profiling

<p>Abstract</p> <p>Background</p> <p>Plant Growth Promoting Rhizobacteria (PGPR), <it>Pseudomonas fluorescens </it>strain KH-1 was found to exhibit plant growth promotional activity in rice under both <it>in-vitro </it>and <it>in-vivo </it>conditions. But the mechanism underlying such promotional activity of <it>P. fluorescens </it>is not yet understood clearly. In this study, efforts were made to elucidate the molecular responses of rice plants to <it>P. fluorescens </it>treatment through protein profiling. Two-dimensional polyacrylamide gel electrophoresis strategy was adopted to identify the PGPR responsive proteins and the differentially expressed proteins were analyzed by mass spectrometry.</p> <p>Results</p> <p>Priming of <it>P. fluorescens</it>, 23 different proteins found to be differentially expressed in rice leaf sheaths and MS analysis revealed the differential expression of some important proteins namely putative p23 co-chaperone, Thioredoxin h- rice, Ribulose-bisphosphate carboxylase large chain precursor, Nucleotide diPhosphate kinase, Proteosome sub unit protein and putative glutathione S-transferase protein.</p> <p>Conclusion</p> <p>Functional analyses of the differential proteins were reported to be directly or indirectly involved in growth promotion in plants. Thus, this study confirms the primary role of PGPR strain KH-1 in rice plant growth promotion.</p

Yersinia pestis intracellular parasitism of macrophages from hosts exhibiting high and low severity of plague.

BACKGROUND: Yersinia pestis causes severe disease in natural rodent hosts, but mild to inapparent disease in certain rodent predators such as dogs. Y. pestis initiates infection in susceptible hosts by parasitizing and multiplying intracellularly in local macrophages prior to systemic dissemination. Thus, we hypothesize that Y. pestis disease severity may depend on the degree to which intracellular Y. pestis overcomes the initial host macrophage imposed stress. METHODOLOGY/PRINCIPAL FINDINGS: To test this hypothesis, the progression of in vitro infection by Y. pestis KIM62053.1+ of mouse splenic and RAW264.7 tissue culture macrophages and dog peripheral blood-derived and DH82 tissue culture macrophages was studied using microscopy and various parameters of infection. The study showed that during the early stage of infection, intracellular Y. pestis assumed filamentous cellular morphology with multiple copies of the genome per bacterium in both mouse and dog macrophages. Later, in mouse macrophages, the infection elicited spacious vacuolar extension of Yersinia containing vacuoles (YCV), and the filamentous Y. pestis reverted to coccobacillary morphology with genomic equivalents approximately equaling colony forming units. In contrast, Y. pestis infected dog macrophages did not show noticeable extension of YCV, and intracellular Y. pestis retained the filamentous cellular morphology for the entire experiment in DH82 cells or were killed by blood-derived macrophages. In addition, during the later stage of infection, Y. pestis infected mouse macrophages exhibited cell lysis whereas dog macrophages did not. CONCLUSION/SIGNIFICANCE: Overall, these results support our hypothesis that Y. pestis in mouse macrophages can overcome the initial intracellular stress necessary for subsequent systemic infection. However, in dogs, failure of Y. pestis to overcome macrophage imposed stress may result in mild or in apparent disease in dogs

Role of Tellurite Resistance Operon in Filamentous Growth of <i>Yersinia pestis</i> in Macrophages

<div><p>Background</p><p><i>Yersinia pestis</i> initiates infection by parasitism of host macrophages. In response to macrophage infections, intracellular <i>Y</i>. <i>pestis</i> can assume a filamentous cellular morphology which may mediate resistance to host cell innate immune responses. We previously observed the expression of <i>Y</i>. <i>pestis</i> tellurite resistance proteins TerD and TerE from the <i>terZABCDE</i> operon during macrophage infections. Others have observed a filamentous response associated with expression of tellurite resistance operon in <i>Escherichia coli</i> exposed to tellurite. Therefore, in this study we examine the potential role of <i>Y</i>. <i>pestis</i> tellurite resistance operon in filamentous cellular morphology during macrophage infections.</p><p>Principal Findings</p><p><i>In vitro</i> treatment of <i>Y</i>. <i>pestis</i> culture with sodium tellurite (Na₂TeO₃) caused the bacterial cells to assume a filamentous phenotype similar to the filamentous phenotype observed during macrophage infections. A deletion mutant for genes <i>terZAB</i> abolished the filamentous morphologic response to tellurite exposure or intracellular parasitism, but without affecting tellurite resistance. However, a <i>terZABCDE</i> deletion mutant abolished both filamentous morphologic response and tellurite resistance. Complementation of the <i>terZABCDE</i> deletion mutant with <i>terCDE</i>, but not <i>terZAB</i>, partially restored tellurite resistance. When the <i>terZABCDE</i> deletion mutant was complemented with <i>terZAB</i> or <i>terCDE</i>, <i>Y</i>. <i>pestis</i> exhibited filamentous morphology during macrophage infections as well as while these complemented genes were being expressed under an <i>in vitro</i> condition. Further in <i>E</i>. <i>coli</i>, expression of <i>Y</i>. <i>pestis terZAB</i>, but not <i>terCDE</i>, conferred a filamentous phenotype.</p><p>Conclusions</p><p>These findings support the role of <i>Y</i>. <i>pestis terZAB</i> mediation of the filamentous response phenotype; whereas, <i>terCDE</i> confers tellurite resistance. Although the beneficial role of filamentous morphological responses by <i>Y</i>. <i>pestis</i> during macrophage infections is yet to be fully defined, it may be a bacterial adaptive strategy to macrophage associated stresses.</p></div

Cellular morphology of <i>Y</i>. <i>pestis</i> KIM6+ <i>ΔterZABCDE</i> complemented with either <i>terZAB</i> or <i>terCDE</i> in extracellular media or during intracellular parasitism.

<p>For panels A-C, <i>Y</i>. <i>pestis</i> KIM6+ <i>ΔterZABCDE</i> mutant transformed with an empty expression plasmid pMMB67EH (A) or complemented with the expression plasmid carrying <i>Y</i>. <i>pestis terZAB</i> (pYP<i>terZAB</i>) (B) or <i>terCDE</i> (pYP<i>terCDE</i>) (C) genes were cultured in RPMI-1640 media containing 1 mM IPTG for A and C, but 0.05 mM IPTG for B. For panels D-F, KIM6+ <i>ΔterZABCDE</i> mutant transformed with an empty expression plasmid pMMB67EH (D) or complemented with the expression plasmid carrying <i>Y</i>. <i>pestis terZAB</i> (pYP<i>terZAB</i>) (E) or <i>terCDE</i> (pYP<i>terCDE</i>) (F) genes infected RAW264.7 cells cultured in 0.05 mM IPTG. Sampled were collected at 2.5 h and observed using Wright Giemsa stain with light microscopy at a magnification of 1,000×.</p

Cellular morphology of <i>Y</i>. <i>pestis</i> KIM6+ <i>ΔterZABCDE</i> complemented with either <i>terZAB</i> or <i>terCDE</i> in extracellular media.

<p><i>Y</i>. <i>pestis</i> KIM6+ <i>ΔterZABCDE</i> mutant transformed with an empty expression plasmid pMMB67EH (A) or complemented with the expression plasmid carrying <i>Y</i>. <i>pestis terZAB</i> (pYP<i>terZAB</i>) (B) or <i>terCDE</i> (pYP<i>terCDE</i>) (C) genes were cultured in RPMI-1640 media without IPTG. Samples were collected at 2.5 h and observed using Wright Giemsa stain with light microscopy at a magnification of 1,000×.</p

Cytotoxicity assay of mouse and dog tissue culture cells infected with <i>Y. pestis</i>.

<p>The specific leakage of the macrophage cytoplasmic enzyme LDH associated with <i>Y. pestis</i> infection of RAW264.7 (•) and DH82 (○) cells was determined. The data are shown as means ± SEM (n = 3). Asterisks on error bars represent, at the respective interval, statistical difference (_*, p<0.05; _**, p<0.01) between the means percentage specific LDH leakage of RAW264.7 and DH82 cells in Student's t-test.</p

Morphological features of intracellular <i>Y. pestis</i> in antibiotic-free media.

<p>RAW264.7 and DH82 cells infected with <i>Y. pestis</i> strain KIM6+ for 30 min in RPMI-1640 with 10% FBS media, washed with PBS to remove extracellular bacteria, and then incubated in HBSS with 10% FBS were sampled at 2.5 and 5 h p.i. for light microscopic examination by staining with Wright Giemsa stain. Arrows indicate filamentous <i>Y. pestis</i>. Images are presented at 1,000× magnification.</p

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p