scholarly journals Spatially Distinct Neutrophil Responses within the Inflammatory Lesions of Pneumonic Plague

mBio ◽  
2015 ◽  
Vol 6 (5) ◽  
Author(s):  
Nikolas M. Stasulli ◽  
Kara R. Eichelberger ◽  
Paul A. Price ◽  
Roger D. Pechous ◽  
Stephanie A. Montgomery ◽  
...  
Keyword(s):  
Yersinia Pestis ◽  
Cell Types ◽  
Lung Lesions ◽  
Pneumonic Plague ◽  
Content Type ◽  
Microbe Interactions ◽  
Host Microbe Interactions ◽  
Neutrophil Survival

ABSTRACTDuring pneumonic plague, the bacteriumYersinia pestiselicits the development of inflammatory lung lesions that continue to expand throughout infection. This lesion development and persistence are poorly understood. Here, we examine spatially distinct regions of lung lesions using laser capture microdissection and transcriptome sequencing (RNA-seq) analysis to identify transcriptional differences between lesion microenvironments. We show that cellular pathways involved in leukocyte migration and apoptosis are downregulated in the center of lung lesions compared to the periphery. Probing for the bacterial factor(s) important for the alteration in neutrophil survival, we show bothin vitroandin vivothatY. pestisincreases neutrophil survival in a manner that is dependent on the type III secretion system effector YopM. This research explores the complexity of spatially distinct host-microbe interactions and emphasizes the importance of cell relevance in assays in order to fully understandY. pestisvirulence.IMPORTANCEYersinia pestisis a high-priority pathogen and continues to cause outbreaks worldwide. The ability ofY. pestisto be transmitted via respiratory droplets and its history of weaponization has led to its classification as a select agent most likely to be used as a biological weapon. Unrestricted bacterial growth during the initial preinflammatory phase primes patients to be infectious once disease symptoms begin in the proinflammatory phase, and the rapid disease progression can lead to death beforeY. pestisinfection can be diagnosed and treated. Usingin vivoanalyses and focusing on relevant cell types during pneumonic plague infection, we can identify host pathways that may be manipulated to extend the treatment window for pneumonic plague patients.

scholarly journals Inactivation of Peroxiredoxin 6 by the Pla Protease of Yersinia pestis

2015 ◽  
Vol 84 (1) ◽  
pp. 365-374 ◽  
Author(s):  
Daniel L. Zimbler ◽  
Justin L. Eddy ◽  
Jay A. Schroeder ◽  
Wyndham W. Lathem
Keyword(s):  
Yersinia Pestis ◽  
Lung Damage ◽  
Rapid Progression ◽  
Dependent Manner ◽  
Wild Type ◽  
Peroxiredoxin 6 ◽  
Pneumonic Plague ◽  
Content Type

Pneumonic plague represents the most severe form of disease caused byYersinia pestisdue to its ease of transmission, rapid progression, and high mortality rate. TheY. pestisouter membrane Pla protease is essential for the development of pneumonic plague; however, the complete repertoire of substrates cleaved by Pla in the lungs is not known. In this study, we describe a proteomic screen to identify host proteins contained within the bronchoalveolar lavage fluid of mice that are cleaved and/or processed byY. pestisin a Pla-dependent manner. We identified peroxiredoxin 6 (Prdx6), a host factor that contributes to pulmonary surfactant metabolism and lung defense against oxidative stress, as a previously unknown substrate of Pla. Pla cleaves Prdx6 at three distinct sites, and these cleavages disrupt both the peroxidase and phospholipase A2activities of Prdx6. In addition, we found that infection with wild-typeY. pestisreduces the abundance of extracellular Prdx6 in the lungs compared to that after infection with ΔplaY. pestis, suggesting that Pla cleaves Prdx6 in the pulmonary compartment. However, following infection with either wild-type or Δpla Y. pestis, Prdx6-deficient mice exhibit no differences in bacterial burden, host immune response, or lung damage from wild-type mice. Thus, while Pla is able to disrupt Prdx6 functionin vitroand reduce Prdx6 levelsin vivo, the cleavage of Prdx6 has little detectable impact on the progression or outcome of pneumonic plague.

scholarly journals Tn-Seq Analysis Identifies Genes Important for Yersinia pestis Adherence during Primary Pneumonic Plague

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Kara R. Eichelberger ◽  
Victoria E. Sepúlveda ◽  
John Ford ◽  
Sara R. Selitsky ◽  
Piotr A. Mieczkowski ◽  
...  
Keyword(s):  
Yersinia Pestis ◽  
Subsequent Development ◽  
Transcriptional Analysis ◽  
Minor Effect ◽  
Wild Type ◽  
Single Strain ◽  
Pneumonic Plague ◽  
Content Type

ABSTRACT Following inhalation, Yersinia pestis rapidly colonizes the lung to establish infection during primary pneumonic plague. Although several adhesins have been identified in Yersinia spp., the factors mediating early Y. pestis adherence in the lung remain unknown. To identify genes important for Y. pestis adherence during primary pneumonic plague, we used transposon insertion sequencing (Tn-seq). Wild-type and capsule mutant (Δcaf1) Y. pestis transposon mutant libraries were serially passaged in vivo to enrich for nonadherent mutants in the lung using a mouse model of primary pneumonic plague. Sequencing of the passaged libraries revealed six mutants that were significantly enriched in both the wild-type and Δcaf1 Y. pestis backgrounds. The enriched mutants had insertions in genes that encode transcriptional regulators, chaperones, an endoribonuclease, and YPO3903, a hypothetical protein. Using single-strain infections and a transcriptional analysis, we identified a significant role for YPO3903 in Y. pestis adherence in the lung and showed that YPO3903 regulated transcript levels of psaA, which encodes a fimbria previously implicated in Y. pestis adherence in vitro. Deletion of psaA had a minor effect on Y. pestis adherence in the lung, suggesting that YPO3903 regulates other adhesins in addition to psaA. By enriching for mutations in genes that regulate the expression or assembly of multiple genes or proteins, we obtained screen results indicating that there may be not just one dominant adhesin but rather several factors that contribute to early Y. pestis adherence during primary pneumonic plague. IMPORTANCE Colonization of the lung by Yersinia pestis is a critical first step in establishing infection during primary pneumonic plague, a disease characterized by high lethality. However, the mechanisms by which Y. pestis adheres in the lung after inhalation remain elusive. Here, we used Tn-seq to identify Y. pestis genes important for adherence early during primary pneumonic plague. Our mutant enrichment strategy resulted in the identification of genes important for regulation and assembly of genes and proteins rather than adhesin genes themselves. These results reveal that there may be multiple Y. pestis adhesins or redundancy among adhesins. Identifying the adhesins regulated by the genes identified in our enrichment screen may reveal novel therapeutic targets for preventing Y. pestis adherence and the subsequent development of pneumonic plague.

scholarly journals Hydroxylamine and Carboxymethoxylamine Can Inhibit Toxoplasma gondii Growth through an Aspartate Aminotransferase-Independent Pathway

2020 ◽  
Vol 64 (3) ◽  
Author(s):  
Jixu Li ◽  
Huanping Guo ◽  
Eloiza May Galon ◽  
Yang Gao ◽  
Seung-Hun Lee ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Drug Targets ◽  
Protozoan Parasite ◽  
Cell Types ◽  
Lytic Cycle ◽  
Type I ◽  
Content Type ◽  
Mechanism Of Inhibition

ABSTRACT Toxoplasma gondii is an obligate intracellular protozoan parasite and a successful parasitic pathogen in diverse organisms and host cell types. Hydroxylamine (HYD) and carboxymethoxylamine (CAR) have been reported as inhibitors of aspartate aminotransferases (AATs) and interfere with the proliferation in Plasmodium falciparum. Therefore, AATs are suggested as drug targets against Plasmodium. The T. gondii genome encodes only one predicted AAT in both T. gondii type I strain RH and type II strain PLK. However, the effects of HYD and CAR, as well as their relationship with AAT, on T. gondii remain unclear. In this study, we found that HYD and CAR impaired the lytic cycle of T. gondii in vitro, including the inhibition of invasion or reinvasion, intracellular replication, and egress. Importantly, HYD and CAR could control acute toxoplasmosis in vivo. Further studies showed that HYD and CAR could inhibit the transamination activity of rTgAAT in vitro. However, our results confirmed that deficiency of AAT in both RH and PLK did not reduce the virulence in mice, although the growth ability of the parasites was affected in vitro. HYD and CAR could still inhibit the growth of AAT-deficient parasites. These findings indicated that HYD and CAR inhibition of T. gondii growth and control of toxoplasmosis can occur in an AAT-independent pathway. Overall, further studies focusing on the elucidation of the mechanism of inhibition are warranted. Our study hints at new substrates of HYD and CAR as potential drug targets to inhibit T. gondii growth.

scholarly journals Upregulation of the Adhesin Gene EPA1 Mediated by PDR1 in Candida glabrata Leads to Enhanced Host Colonization

mSphere ◽  
2016 ◽  
Vol 1 (2) ◽  
Author(s):  
Luis A. Vale-Silva ◽  
Beat Moeckli ◽  
Riccardo Torelli ◽  
Brunella Posteraro ◽  
Maurizio Sanguinetti ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Epithelial Cells ◽  
Candida Glabrata ◽  
Resistance Mechanism ◽  
Cell Types ◽  
Host Cells ◽  
Regulation Mechanism ◽  
Content Type

ABSTRACT Candida glabrata is an important fungal pathogen in human diseases and is also rapidly acquiring drug resistance. Drug resistance can be mediated by the transcriptional activator PDR1, and this results in the upregulation of multidrug transporters. Intriguingly, this resistance mechanism is associated in C. glabrata with increased virulence in animal models and also with increased adherence to specific host cell types. The C. glabrata adhesin gene EPA1 is a major contributor of virulence and adherence to host cells. Here, we show that EPA1 expression is controlled by PDR1 independently of subtelomeric silencing, a known EPA1 regulation mechanism. Thus, a relationship exists between PDR1, EPA1 expression, and adherence to host cells, which is critical for efficient virulence. Our results demonstrate that acquisition of drug resistance is beneficial for C. glabrata in fungus-host relationships. These findings further highlight the challenges of the therapeutic management of C. glabrata infections in human patients. Candida glabrata is the second most common Candida species causing disseminated infection, after C. albicans. C. glabrata is intrinsically less susceptible to the widely used azole antifungal drugs and quickly develops secondary resistance. Resistance typically relies on drug efflux with transporters regulated by the transcription factor Pdr1. Gain-of-function (GOF) mutations in PDR1 lead to a hyperactive state and thus efflux transporter upregulation. Our laboratory has characterized a collection of C. glabrata clinical isolates in which azole resistance was found to correlate with increased virulence in vivo. Contributing phenotypes were the evasion of adhesion and phagocytosis by macrophages and an increased adhesion to epithelial cells. These phenotypes were found to be dependent on PDR1 GOF mutation and/or C. glabrata strain background. In the search for the molecular effectors, we found that PDR1 hyperactivity leads to overexpression of specific cell wall adhesins of C. glabrata. Further study revealed that EPA1 regulation, in particular, explained the increase in adherence to epithelial cells. Deleting EPA1 eliminates the increase in adherence in an in vitro model of interaction with epithelial cells. In a murine model of urinary tract infection, PDR1 hyperactivity conferred increased ability to colonize the bladder and kidneys in an EPA1-dependent way. In conclusion, this study establishes a relationship between PDR1 and the regulation of cell wall adhesins, an important virulence attribute of C. glabrata. Furthermore, our data show that PDR1 hyperactivity mediates increased adherence to host epithelial tissues both in vitro and in vivo through upregulation of the adhesin gene EPA1. IMPORTANCE Candida glabrata is an important fungal pathogen in human diseases and is also rapidly acquiring drug resistance. Drug resistance can be mediated by the transcriptional activator PDR1, and this results in the upregulation of multidrug transporters. Intriguingly, this resistance mechanism is associated in C. glabrata with increased virulence in animal models and also with increased adherence to specific host cell types. The C. glabrata adhesin gene EPA1 is a major contributor of virulence and adherence to host cells. Here, we show that EPA1 expression is controlled by PDR1 independently of subtelomeric silencing, a known EPA1 regulation mechanism. Thus, a relationship exists between PDR1, EPA1 expression, and adherence to host cells, which is critical for efficient virulence. Our results demonstrate that acquisition of drug resistance is beneficial for C. glabrata in fungus-host relationships. These findings further highlight the challenges of the therapeutic management of C. glabrata infections in human patients.

scholarly journals Dolosigranulum pigrum Cooperation and Competition in Human Nasal Microbiota

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Silvio D. Brugger ◽  
Sara M. Eslami ◽  
Melinda M. Pettigrew ◽  
Isabel F. Escapa ◽  
Matthew T. Henke ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Streptococcus Pneumoniae ◽  
Genomic Analysis ◽  
Upper Respiratory Tract ◽  
Content Type ◽  
Microbe Interactions ◽  
Clinical Experiments ◽  
Nasal Microbiota

ABSTRACT Multiple epidemiological studies identify Dolosigranulum pigrum as a candidate beneficial bacterium based on its positive association with health, including negative associations with nasal/nasopharyngeal colonization by the pathogenic species Staphylococcus aureus and Streptococcus pneumoniae. Using a multipronged approach to gain new insights into D. pigrum function, we observed phenotypic interactions and predictions of genomic capacity that support the idea of a role for microbe-microbe interactions involving D. pigrum in shaping the composition of human nasal microbiota. We identified in vivo community-level and in vitro phenotypic cooperation by specific nasal Corynebacterium species. Also, D. pigrum inhibited S. aureus growth in vitro, whereas robust inhibition of S. pneumoniae required both D. pigrum and a nasal Corynebacterium together. D. pigrum l-lactic acid production was insufficient to account for these inhibitions. Genomic analysis of 11 strains revealed that D. pigrum has a small genome (average 1.86 Mb) and multiple predicted auxotrophies consistent with D. pigrum relying on its human host and on cocolonizing bacteria for key nutrients. Further, the accessory genome of D. pigrum harbored a diverse repertoire of biosynthetic gene clusters, some of which may have a role in microbe-microbe interactions. These new insights into D. pigrum’s functions advance the field from compositional analysis to genomic and phenotypic experimentation on a potentially beneficial bacterial resident of the human upper respiratory tract and lay the foundation for future animal and clinical experiments. IMPORTANCE Staphylococcus aureus and Streptococcus pneumoniae infections cause significant morbidity and mortality in humans. For both, nasal colonization is a risk factor for infection. Studies of nasal microbiota identify Dolosigranulum pigrum as a benign bacterium present when adults are free of S. aureus or when children are free of S. pneumoniae. Here, we validated these in vivo associations with functional assays. We found that D. pigrum inhibited S. aureus in vitro and, together with a specific nasal Corynebacterium species, also inhibited S. pneumoniae. Furthermore, genomic analysis of D. pigrum indicated that it must obtain key nutrients from other nasal bacteria or from humans. These phenotypic interactions support the idea of a role for microbe-microbe interactions in shaping the composition of human nasal microbiota and implicate D. pigrum as a mutualist of humans. These findings support the feasibility of future development of microbe-targeted interventions to reshape nasal microbiota composition to exclude S. aureus and/or S. pneumoniae.

scholarly journals Tissue Distribution of Doxycycline in Animal Models of Tuberculosis

2020 ◽  
Vol 64 (5) ◽  
Author(s):  
Martin Gengenbacher ◽  
Matthew D. Zimmerman ◽  
Jansy P. Sarathy ◽  
Firat Kaya ◽  
Han Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptional Control ◽  
Target Genes ◽  
Calcium Content ◽  
Tissue Penetration ◽  
Lung Lesions ◽  
Content Type ◽  
Clearance Kinetics

ABSTRACT Doxycycline, an FDA-approved tetracycline, is used in tuberculosis in vivo models for the temporal control of mycobacterial gene expression. In these models, animals are infected with recombinant Mycobacterium tuberculosis carrying genes of interest under transcriptional control of the doxycycline-responsive TetR-tetO unit. To minimize fluctuations of plasma levels, doxycycline is usually administered in the diet. However, tissue penetration studies to identify the minimum doxycycline content in food achieving complete repression of TetR-controlled genes in tuberculosis (TB)-infected organs and lesions have not been conducted. Here, we first determined the tetracycline concentrations required to achieve silencing of M. tuberculosis target genes in vitro. Next, we measured doxycycline concentrations in plasma, major organs, and lung lesions in TB-infected mice and rabbits and compared these values to silencing concentrations measured in vitro. We found that 2,000 ppm doxycycline supplemented in mouse and rabbit feed is sufficient to reach target concentrations in TB lesions. In rabbit chow, the calcium content had to be reduced 5-fold to minimize chelation of doxycycline and deliver adequate oral bioavailability. Clearance kinetics from major organs and lung lesions revealed that doxycycline levels fall below concentrations that repress tet promoters within 7 to 14 days after doxycycline is removed from the diet. In summary, we have shown that 2,000 ppm doxycycline supplemented in standard mouse diet and in low-calcium rabbit diet delivers concentrations adequate to achieve full repression of tet promoters in infected tissues of mice and rabbits.

scholarly journals Effects of Psa and F1 on the Adhesive and Invasive Interactions of Yersinia pestis with Human Respiratory Tract Epithelial Cells

2006 ◽  
Vol 74 (10) ◽  
pp. 5636-5644 ◽  
Author(s):  
Fengzhi Liu ◽  
Huaiqing Chen ◽  
Estela M. Galván ◽  
Melissa A. Lasaro ◽  
Dieter M. Schifferli
Keyword(s):  
Epithelial Cells ◽  
Respiratory Tract ◽  
Yersinia Pestis ◽  
Bacterial Adhesion ◽  
Wild Type Strain ◽  
Cell Types ◽  
Human Respiratory Tract ◽  
Bacterial Uptake

ABSTRACT Yersinia pestis, the causative agent of plague, expresses the Psa fimbriae (pH 6 antigen) in vitro and in vivo. To evaluate the potential virulence properties of Psa for pneumonic plague, an Escherichia coli strain expressing Psa was engineered and shown to adhere to three types of human respiratory tract epithelial cells. Psa binding specificity was confirmed with Psa-coated polystyrene beads and by inhibition assays. Individual Y. pestis cells were found to be able to express the capsular antigen fraction 1 (F1) concomitantly with Psa on their surface when analyzed by flow cytometry. To better evaluate the separate effects of F1 and Psa on the adhesive and invasive properties of Y. pestis, isogenic Δcaf (F1 genes), Δpsa, and Δcaf Δpsa mutants were constructed and studied with the three respiratory tract epithelial cells. The Δpsa mutant bound significantly less to all three epithelial cells compared to the parental wild-type strain and the Δcaf and Δcaf Δpsa mutants, indicating that Psa acts as an adhesin for respiratory tract epithelial cells. An antiadhesive effect of F1 was clearly detectable only in the absence of Psa, underlining the dominance of the Psa+ phenotype. Both F1 and Psa inhibited the intracellular uptake of Y. pestis. Thus, F1 inhibits bacterial uptake by inhibiting bacterial adhesion to epithelial cells, whereas Psa seems to block bacterial uptake by interacting with a host receptor that doesn't direct internalization. The Δcaf Δpsa double mutant bound and invaded all three epithelial cell types well, revealing the presence of an undefined adhesin(s) and invasin(s).

scholarly journals Gut mélange à trois: fluctuating selection modulated by microbiota, host immune system, and antibiotics

2021 ◽  
Author(s):  
Hugo Condessa Barreto ◽  
Beatriz Abreu ◽  
Isabel Gordo
Keyword(s):  
Immune System ◽  
Iron Homeostasis ◽  
Host Immune System ◽  
Iron Availability ◽  
Lipocalin 2 ◽  
Fluctuating Selection ◽  
Microbe Interactions ◽  
Host Microbe Interactions

Iron is critical in host-microbe interactions, and its availability is under tight regulation in the mammalian gut. Antibiotics and inflammation are known to perturb iron availability in the gut, which could subsequently alter host-microbe interactions. Here, we show that an adaptive allele of iscR, encoding a major regulator of iron homeostasis of Escherichia coli, is under fluctuating selection in the mouse gut. In vivo competitions in immune-competent, immune-compromised, and germ-free mice reveal that the selective pressure on an iscR mutant E. coli is modulated by the presence of antibiotics, other members of the microbiota, and the immune system. In vitro assays show that iron availability is an important mediator of the iscR allele fitness benefits or costs. We identify Lipocalin-2, a host's innate immune system protein that prevents bacterial iron acquisition, as a major host mechanism underlying fluctuating selection of the iscR allele. Our results provide a remarkable example of strong fluctuating selection acting on bacterial iron regulation in the mammalian gut.

scholarly journals In Vivo Transcriptional Profiling of Yersinia pestis Reveals a Novel Bacterial Mediator of Pulmonary Inflammation

mBio ◽  
2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Roger D. Pechous ◽  
Christopher A. Broberg ◽  
Nikolas M. Stasulli ◽  
Virginia L. Miller ◽  
William E. Goldman
Keyword(s):  
Yersinia Pestis ◽  
Inflammatory Responses ◽  
Severe Pneumonia ◽  
Sudden Onset ◽  
Host Factors ◽  
Respiratory Pathogen ◽  
Bacterial Survival ◽  
Pneumonic Plague ◽  
Content Type

ABSTRACTInhalation ofYersinia pestisresults in primary pneumonic plague, a highly lethal and rapidly progressing necrotizing pneumonia. The disease begins with a period of extensive bacterial replication in the absence of disease symptoms, followed by the sudden onset of inflammatory responses that ultimately prove fatal. Very little is known about the bacterial and host factors that contribute to the rapid biphasic progression of pneumonic plague. In this work, we analyzed thein vivotranscription kinetics of 288 bacterial open reading frames previously shown by microarray analysis to be dynamically regulated in the lung. Using this approach combined with bacterial genetics, we were able to identify five Y. pestis genes that contribute to the development of pneumonic plague. Deletion of one of these genes,ybtX, did not alter bacterial survival but attenuated host inflammatory responses during late-stage disease. Deletion ofybtXin another lethal respiratory pathogen,Klebsiella pneumoniae, also resulted in diminished host inflammation during infection. Thus, ourin vivotranscriptional screen has identified an important inflammatory mediator that is common to two Gram-negative bacterial pathogens that cause severe pneumonia.IMPORTANCEYersinia pestis is responsible for at least three major pandemics, most notably the Black Death of the Middle Ages. Due to its pandemic potential, ease of dissemination by aerosolization, and a history of its weaponization, Y. pestis is categorized by the Centers for Disease Control and Prevention as a tier 1 select agent most likely to be used as a biological weapon. To date, there is no licensed vaccine against Y. pestis. Importantly, an early “silent” phase followed by the rapid onset of nondescript influenza-like symptoms makes timely treatment of pneumonic plague difficult. A more detailed understanding of the bacterial and host factors that contribute to pathogenesis is essential to understanding the progression of pneumonic plague and developing or enhancing treatment options.

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