Depletion of Glucose Activates Catabolite Repression during Pneumonic Plague.

Bacterial pathogenesis depends on changes in metabolic and virulence gene expression in response to changes within a pathogen's environment. The plague-causing pathogen, , requires expression of the gene encoding the Pla protease for progression of pneumonic plague. The catabolite repressor protein Crp, a global transcriptional regulator, may serve as the activator of in response to changes within the lungs as disease progresses. By using gene reporter fusions, the spatial and temporal activation of the and promoters was measured in a mouse model of pneumonic plague. In the lungs, was highly expressed in bacteria found within large aggregates resembling biofilms, while expression increased over time independent of the aggregated state. Increased expression of and correlated with a reduction in lung glucose levels. Deletion of the glucose-specific phosphotransferase system EIIBC (PtsG) of rescued glucose levels in the lungs, resulting in reduced expression of both and We propose that activation of expression during pneumonic plague is driven by an increase of both Crp and cAMP levels following consumption of available glucose in the lungs by Thus, Crp operates as a sensor linking the nutritional environment of the host to regulation of virulence gene expression. Using as a model for pneumonia, we discovered that glucose is rapidly consumed, leading to a catabolite-repressive environment in the lungs. As a result, expression of the gene encoding the plasminogen activator protease, a target of the catabolite repressor protein required for pathogenesis, is activated. Interestingly, expression of the catabolite repressor protein itself was also increased in the absence of glucose but only in biofilms. The data presented here demonstrate how a bacterial pathogen senses changes within its environment to coordinate metabolism and virulence gene expression.

Ritzert JT ，Lathem WW 《-》

Proteolysis of plasminogen activator inhibitor-1 by Yersinia pestis remodulates the host environment to promote virulence.

Essentials Effect of plasminogen activator inhibitor (PAI)-1 on plague and its Y. pestis cleavage is unknown. An intranasal mouse model of infection was used to determine the role of PAI-1 in pneumonic plague. PAI-1 is cleaved and inactivated by the Pla protease of Y. pestis in the lung airspace. PAI-1 impacts both bacterial outgrowth and the immune response to respiratory Y. pestis infection. Click to hear Dr Bock discuss pathogen activators of plasminogen. Background The hemostatic regulator plasminogen activator inhibitor-1 (PAI-1) inactivates endogenous plasminogen activators and aids in the immune response to bacterial infection. Yersinia pestis, the causative agent of plague, produces the Pla protease, a virulence factor that is required during plague. However, the specific hemostatic proteins cleaved by Pla in vivo that contribute to pathogenesis have not yet been fully elucidated. Objectives To determine whether PAI-1 is cleaved by the Pla protease during pneumonic plague, and to define the impact of PAI-1 on Y. pestis respiratory infection in the presence or absence of Pla. Methods An intranasal mouse model of pneumonic plague was used to assess the levels of total and active PAI-1 in the lung airspace, and the impact of PAI-1 deficiency on bacterial pathogenesis, the host immune response and plasmin generation following infection with wild-type or ∆pla Y. pestis. Results We found that Y. pestis cleaves and inactivates PAI-1 in the lungs in a Pla-dependent manner. The loss of PAI-1 enhances Y. pestis outgrowth in the absence of Pla, and is associated with increased conversion of plasminogen to plasmin. Furthermore, we found that PAI-1 regulates immune cell recruitment, cytokine production and tissue permeability during pneumonic plague. Conclusions Our data demonstrate that PAI-1 is an in vivo target of the Pla protease in the lungs, and that PAI-1 is a key regulator of the pulmonary innate immune response. We conclude that the inactivation of PAI-1 by Y. pestis alters the host environment to promote virulence during pneumonic plague.

Eddy JL ，Schroeder JA ，Zimbler DL ，Caulfield AJ ，Lathem WW ... -  《-》

The Cyclic AMP Receptor Protein Regulates Quorum Sensing and Global Gene Expression in Yersinia pestis during Planktonic Growth and Growth in Biofilms.

Cyclic AMP (cAMP) receptor protein (Crp) is an important transcriptional regulator of Expression of increases during pneumonic plague as the pathogen depletes glucose and forms large biofilms within lungs. To better understand control of Crp, we determined a 1.8-Å crystal structure of the protein-cAMP complex. We found that compared to Crp, C helix amino acid substitutions in Crp did not impact the cAMP dependency of Crp to bind DNA promoters. To investigate Crp-regulated genes during plague pneumonia, we performed RNA sequencing on both wild-type and Δ mutant bacteria growing in planktonic and biofilm states in minimal media with glucose or glycerol. Crp was found to dramatically alter expression of hundreds of genes in a manner dependent upon carbon source and growth state. Gel shift assays confirmed direct regulation of the and promoters, and Crp was then linked to growth on maltose as a sole carbon source. Iron regulation genes and were found to be indirectly regulated by Crp. A new connection between carbon source and quorum sensing was revealed as Crp was found to regulate production of acyl-homoserine lactones (AHLs) through direct and indirect regulation of genes for AHL synthetases and receptors. AHLs were subsequently identified in the lungs of -infected mice when expression was highest in biofilms. Thus, in addition to the well-studied gene, other Crp-regulated genes likely have important functions during plague infection. Bacterial pathogens have evolved extensive signaling pathways to translate environmental signals into changes in gene expression. While Crp has long been appreciated for its role in regulating metabolism of carbon sources in many bacterial species, transcriptional profiling has revealed that this protein regulates many other aspects of bacterial physiology. The plague pathogen requires this global regulator to survive in blood, skin, and lungs. During disease progression, this organism adapts to changes within these niches. In addition to regulating genes for metabolism of nonglucose sugars, we found that Crp regulates genes for virulence, metal acquisition, and quorum sensing by direct or indirect mechanisms. Thus, this single transcriptional regulator, which responds to changes in available carbon sources, can regulate multiple critical behaviors for causing disease.

Ritzert JT ，Minasov G ，Embry R ，Schipma MJ ，Satchell KJF ... -  《mBio》

Posttranscriptional regulation of the Yersinia pestis cyclic AMP receptor protein Crp and impact on virulence.

The cyclic AMP receptor protein (Crp) is a transcriptional regulator that controls the expression of numerous bacterial genes, usually in response to environmental conditions and particularly by sensing the availability of carbon. In the plague pathogen Yersinia pestis, Crp regulates the expression of multiple virulence factors, including components of the type III secretion system and the plasminogen activator protease Pla. The regulation of Crp itself, however, is distinctly different from that found in the well-studied Escherichia coli system. Here, we show that at physiological temperatures, the synthesis of Crp in Y. pestis is positively regulated at the posttranscriptional level. The loss of the small RNA chaperone Hfq results in decreased Crp protein levels but not in steady-state Crp transcript levels, and this regulatory effect occurs within the 5' untranslated region (UTR) of the Crp mRNA. The posttranscriptional activation of Crp synthesis is required for the expression of pla, and decoupling crp from Hfq through the use of an exogenously controlled promoter and 5' UTR increases Pla protein levels as well as partially rescues the growth defect associated with the loss of Hfq. Finally, we show that both Hfq and the posttranscriptional regulation of Crp contribute to the virulence of Y. pestis during pneumonic plague. The Hfq-dependent, posttranscriptional regulation of Crp may be specific to Yersinia species, and thus our data help explain the dramatic growth and virulence defects associated with the loss of Hfq in Y. pestis. The Crp protein is a major transcriptional regulator in bacteria, and its synthesis is tightly controlled to avoid inappropriate induction of the Crp regulon. In this report, we provide the first evidence of Crp regulation in an Hfq-dependent manner at the posttranscriptional level. Our discovery that the synthesis of Crp in Yersinia pestis is Hfq dependent adds an additional layer of regulation to catabolite repression in this bacterium. Our work provides a mechanism by which the plague pathogen links not just the sensing of glucose or other carbon sources but also other signals that influence Crp abundance via the expression of small RNAs to the induction of the Crp regulon. In turn, this allows Y. pestis to fine-tune Crp levels to optimize virulence gene expression during plague infection and may allow the bacterium to adapt to its unique environmental niches.

Lathem WW ，Schroeder JA ，Bellows LE ，Ritzert JT ，Koo JT ，Price PA ，Caulfield AJ ，Goldman WE ... -  《mBio》

A plasminogen-activating protease specifically controls the development of primary pneumonic plague.

Primary pneumonic plague is transmitted easily, progresses rapidly, and causes high mortality, but the mechanisms by which Yersinia pestis overwhelms the lungs are largely unknown. We show that the plasminogen activator Pla is essential for Y. pestis to cause primary pneumonic plague but is less important for dissemination during pneumonic plague than during bubonic plague. Experiments manipulating its temporal expression showed that Pla allows Y. pestis to replicate rapidly in the airways, causing a lethal fulminant pneumonia; if unexpressed, inflammation is aborted, and lung repair is activated. Inhibition of Pla expression prolonged the survival of animals with the disease, offering a therapeutic option to extend the period during which antibiotics are effective.

Lathem WW ，Price PA ，Miller VL ，Goldman WE ... -  《-》