scholarly journals Microevolution During Serial Mouse Passage Demonstrates FRE3 as a Virulence Adaptation Gene in Cryptococcus neoformans

mBio ◽  
2014 ◽  
Vol 5 (2) ◽  
Author(s):  
Guowu Hu ◽  
Shu Hui Chen ◽  
Jin Qiu ◽  
John E. Bennett ◽  
Timothy G. Myers ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Transcriptome Sequencing ◽  
Molecular Mechanisms ◽  
Reductase Activity ◽  
The United States ◽  
Microbial Pathogens ◽  
Content Type ◽  
Time To Death ◽  
Expression Studies ◽  
Iron Reductase

ABSTRACTPassage in mice of opportunistic pathogens such asCryptococcus neoformansis known to increase virulence, but little is known about the molecular mechanisms involved in virulence adaptation. Serial mouse passage of nine environmental strains of serotype AC. neoformansidentified two highly adapted virulent strains that showed a 4-fold reduction in time to death after four passages. Transcriptome sequencing expression studies demonstrated increased expression of aFRE3-encoded iron reductase in the two strains but not in a control strain that did not demonstrate increased virulence during mouse passage.FRE3was shown to express an iron reductase activity and to play a role in iron-dependent growth ofC. neoformans. Overexpression ofFRE3in the two original environmental strains increased growth in the macrophage cell line J774.16 and increased virulence. These data demonstrate a role forFRE3in the virulence ofC. neoformansand demonstrate how the increased expression of such a “virulence acquisition gene” during the environment-to-mammal transition, can optimize the virulence of environmental strains in mammalian hosts.IMPORTANCECryptococcus neoformansis a significant global fungal pathogen that also resides in the environment. Recent studies have suggested that the organism may undergo microevolution in the host. However, little is known about the permitted genetic changes facilitating the adaptation of environmental strains to mammalian hosts. The present studies subjected environmental strains isolated from several metropolitan areas of the United States to serial passages in mice. Transcriptome sequencing expression studies identified the increased expression of an iron reductase gene,FRE3, in two strains that adapted in mice to become highly virulent, and overexpression ofFRE3recapitulated the increased virulence after mouse passage. Iron reductase in yeast is important to iron uptake in a large number of microbial pathogens. These studies demonstrate the capacity ofC. neoformansto show reproducible changes in the expression levels of small numbers of genes termed “virulence adaptation genes” to effectively increase pathogenicity during the environment-to-mammal transition.

scholarly journals DAP12 Inhibits Pulmonary Immune Responses to Cryptococcus neoformans

2016 ◽  
Vol 84 (6) ◽  
pp. 1879-1886 ◽  
Author(s):  
Lena J. Heung ◽  
Tobias M. Hohl
Keyword(s):  
Immune Response ◽  
Nk Cells ◽  
Cryptococcus Neoformans ◽  
Molecular Mechanisms ◽  
Content Type ◽  
Effector Cells ◽  
Opportunistic Fungal Pathogen ◽  
Efficient Uptake ◽  
Bacteria And Fungi

Cryptococcus neoformansis an opportunistic fungal pathogen that is inhaled into the lungs and can lead to life-threatening meningoencephalitis in immunocompromised patients. Currently, the molecular mechanisms that regulate the mammalian immune response to respiratory cryptococcal challenge remain poorly defined. DAP12, a signaling adapter for multiple pattern recognition receptors in myeloid and natural killer (NK) cells, has been shown to play both activating and inhibitory roles during lung infections by different bacteria and fungi. In this study, we demonstrate that DAP12 plays an important inhibitory role in the immune response toC. neoformans. Infectious outcomes in DAP12−/−mice, including survival and lung fungal burden, are significantly improved compared to those in C57BL/6 wild-type (WT) mice. We find that eosinophils and macrophages are decreased while NK cells are increased in the lungs of infected DAP12−/−mice. In contrast to WT NK cells, DAP12−/−NK cells are able to repressC. neoformansgrowthin vitro. Additionally, DAP12−/−macrophages are more highly activated than WT macrophages, with increased production of tumor necrosis factor (TNF) and CCL5/RANTES and more efficient uptake and killing ofC. neoformans. These findings suggest that DAP12 acts as a brake on the pulmonary immune response toC. neoformansby promoting pulmonary eosinophilia and by inhibiting the activation and antifungal activities of effector cells, including NK cells and macrophages.

scholarly journals Global Dissemination ofblaKPCinto Bacterial Species beyond Klebsiella pneumoniae andIn VitroSusceptibility to Ceftazidime-Avibactam and Aztreonam-Avibactam

2016 ◽  
Vol 60 (8) ◽  
pp. 4490-4500 ◽  
Author(s):  
Krystyna M. Kazmierczak ◽  
Douglas J. Biedenbach ◽  
Meredith Hackel ◽  
Sharon Rabine ◽  
Boudewijn L. M. de Jonge ◽  
...  
Keyword(s):  
United States ◽  
Klebsiella Pneumoniae ◽  
Molecular Mechanisms ◽  
Bacterial Species ◽  
The United States ◽  
Asia Pacific ◽  
Gram Negative ◽  
Content Type ◽  
Global Problem

ABSTRACTTheKlebsiella pneumoniaecarbapenemase (KPC), first described in the United States in 1996, is now a widespread global problem in several Gram-negative species. A worldwide surveillance study collected Gram-negative pathogens from 202 global sites in 40 countries during 2012 to 2014 and determined susceptibility to β-lactams and other class agents by broth microdilution testing. Molecular mechanisms of β-lactam resistance among carbapenem-nonsusceptibleEnterobacteriaceaeandPseudomonas aeruginosawere determined using PCR and sequencing. Genes encoding KPC enzymes were found in 586 isolates from 22 countries (76 medical centers), including countries in the Asia-Pacific region (32 isolates), Europe (264 isolates), Latin America (210 isolates), and the Middle East (19 isolates, Israel only) and the United States (61 isolates). The majority of isolates wereK. pneumoniae(83.4%); however, KPC was detected in 13 additional species. KPC-2 (69.6%) was more common than KPC-3 (29.5%), with regional variation observed. A novel KPC variant, KPC-18 (KPC-3[V8I]), was identified during the study. Few antimicrobial agents tested remained effectivein vitroagainst KPC-producing isolates, with ceftazidime-avibactam (MIC90, 4 μg/ml), aztreonam-avibactam (MIC90, 0.5 μg/ml), and tigecycline (MIC90, 2 μg/ml) retaining the greatest activity againstEnterobacteriaceaecocarrying KPC and other β-lactamases, whereas colistin (MIC90, 2 μg/ml) demonstrated the greatestin vitroactivity against KPC-positiveP. aeruginosa. This analysis of surveillance data demonstrated that KPC is widely disseminated. KPC was found in multiple species ofEnterobacteriaceaeandP. aeruginosaand has now become a global problem.

scholarly journals A Prolonged Outbreak of KPC-3-Producing Enterobacter cloacae and Klebsiella pneumoniae Driven by Multiple Mechanisms of Resistance Transmission at a Large Academic Burn Center

2016 ◽  
Vol 61 (2) ◽  
Author(s):  
Hajime Kanamori ◽  
Christian M. Parobek ◽  
Jonathan J. Juliano ◽  
David van Duin ◽  
Bruce A. Cairns ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Molecular Mechanisms ◽  
Enterobacter Cloacae ◽  
The United States ◽  
Multidrug Resistant ◽  
Control Measures ◽  
Content Type ◽  
Burn Center ◽  
Infection Control Measures ◽  
Carbapenem Resistant

ABSTRACT Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacter cloacae has been recently recognized in the United States. Whole-genome sequencing (WGS) has become a useful tool for analysis of outbreaks and for determining transmission networks of multidrug-resistant organisms in health care settings, including carbapenem-resistant Enterobacteriaceae (CRE). We experienced a prolonged outbreak of CRE E. cloacae and K. pneumoniae over a 3-year period at a large academic burn center despite rigorous infection control measures. To understand the molecular mechanisms that sustained this outbreak, we investigated the CRE outbreak isolates by using WGS. Twenty-two clinical isolates of CRE, including E. cloacae (n = 15) and K. pneumoniae (n = 7), were sequenced and analyzed genetically. WGS revealed that this outbreak, which seemed epidemiologically unlinked, was in fact genetically linked over a prolonged period. Multiple mechanisms were found to account for the ongoing outbreak of KPC-3-producing E. cloacae and K. pneumoniae. This outbreak was primarily maintained by a clonal expansion of E. cloacae sequence type 114 (ST114) with distribution of multiple resistance determinants. Plasmid and transposon analyses suggested that the majority of bla KPC-3 was transmitted via an identical Tn4401b element on part of a common plasmid. WGS analysis demonstrated complex transmission dynamics within the burn center at levels of the strain and/or plasmid in association with a transposon, highlighting the versatility of KPC-producing Enterobacteriaceae in their ability to utilize multiple modes to resistance gene propagation.

scholarly journals Structural Biology of the Arterivirus nsp11 Endoribonucleases

2016 ◽  
Vol 91 (1) ◽  
Author(s):  
Manfeng Zhang ◽  
Xiaorong Li ◽  
Zengqin Deng ◽  
Zhenhang Chen ◽  
Yang Liu ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Nonstructural Protein ◽  
Antiviral Drug ◽  
The United States ◽  
Equine Arteritis Virus ◽  
Respiratory Syndrome Virus ◽  
Human Pathogens ◽  
Content Type ◽  
Terminal Domain ◽  
Insight Into

ABSTRACT Endoribonuclease (NendoU) is unique and conserved as a major genetic marker in nidoviruses that infect vertebrate hosts. Arterivirus nonstructural protein 11 (nsp11) was shown to have NendoU activity and play essential roles in the viral life cycle. Here, we report three crystal structures of porcine reproductive and respiratory syndrome virus (PRRSV) and equine arteritis virus (EAV) nsp11 mutants. The structures of arterivirus nsp11 contain two conserved compact domains: the N-terminal domain (NTD) and C-terminal domain (CTD). The structures of PRRSV and EAV endoribonucleases are similar and conserved in the arterivirus, but they are greatly different from that of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses (CoV), representing important human pathogens in the Nidovirales order. The catalytic center of NendoU activity is located in the CTD, where a positively charged groove is next to the key catalytic residues conserved in nidoviruses. Although the NTD is nearly identical, the catalytic region of the arterivirus nsp11 family proteins is remarkably flexible, and the oligomerization may be concentration dependent. In summary, our structures provide new insight into this key multifunctional NendoU family of proteins and lay a foundation for better understanding of the molecular mechanism and antiviral drug development. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) and equine arteritis virus are two major members of the arterivirus family. PRRSV, a leading swine pathogen, causes reproductive failure in breeding stock and respiratory tract illness in young pigs. Due to the lack of a suitable vaccine or effective drug treatment and the quick spread of these viruses, infected animals either die quickly or must be culled. PRRSV costs the swine industry around $644 million annually in the United States and almost €1.5 billion in Europe every year. To find a way to combat these viruses, we focused on the essential viral nonstructural protein 11 (nsp11). nsp11 is associated with multiple functions, such as RNA processing and suppression of the infected host innate immunity system. The three structures solved in this study provide new insight into the molecular mechanisms of this crucial protein family and will benefit the development of new treatments against these deadly viruses.

scholarly journals CmeABC Multidrug Efflux Pump Contributes to Antibiotic Resistance and Promotes Campylobacter jejuni Survival and Multiplication in Acanthamoeba polyphaga

2017 ◽  
Vol 83 (22) ◽  
Author(s):  
Ana Vieira ◽  
Amritha Ramesh ◽  
Alan M. Seddon ◽  
Andrey V. Karlyshev
Keyword(s):  
Antibiotic Resistance ◽  
Campylobacter Jejuni ◽  
Molecular Mechanisms ◽  
Bacterial Resistance ◽  
Efflux Pump ◽  
Synergistic Effects ◽  
Microbial Pathogens ◽  
Bacterial Survival ◽  
Acanthamoeba Polyphaga ◽  
Content Type

ABSTRACT Campylobacter jejuni is a foodborne pathogen that is recognized as the leading cause of human bacterial gastroenteritis. The widespread use of antibiotics in medicine and in animal husbandry has led to an increased incidence of antibiotic resistance in Campylobacter. In addition to a role in multidrug resistance (MDR), the Campylobacter CmeABC resistance-nodulation-division (RND)-type efflux pump may be involved in virulence. As a vehicle for pathogenic microorganisms, the protozoan Acanthamoeba is a good model for investigations of bacterial survival in the environment and the molecular mechanisms of pathogenicity. The interaction between C. jejuni 81-176 and Acanthamoeba polyphaga was investigated in this study by using a modified gentamicin protection assay. In addition, a possible role for the CmeABC MDR pump in this interaction was explored. Here we report that this MDR pump is beneficial for the intracellular survival and multiplication of C. jejuni in A. polyphaga but is dispensable for biofilm formation and motility. IMPORTANCE The endosymbiotic relationship between amoebae and microbial pathogens may contribute to persistence and spreading of the latter in the environment, which has significant implications for human health. In this study, we found that Campylobacter jejuni was able to survive and to multiply inside Acanthamoeba polyphaga; since these microorganisms can coexist in the same environment (e.g., on poultry farms), the latter may increase the risk of infection with Campylobacter. Our data suggest that, in addition to its role in antibiotic resistance, the CmeABC MDR efflux pump plays a role in bacterial survival within amoebae. Furthermore, we demonstrated synergistic effects of the CmeABC MDR efflux pump and TetO on bacterial resistance to tetracycline. Due to its role in both the antibiotic resistance and the virulence of C. jejuni, the CmeABC MDR efflux pump could be considered a good target for the development of antibacterial drugs against this pathogen.

scholarly journals International Evaluation of MIC Distributions and Epidemiological Cutoff Value (ECV) Definitions for Fusarium Species Identified by Molecular Methods for the CLSI Broth Microdilution Method

2015 ◽  
Vol 60 (2) ◽  
pp. 1079-1084 ◽  
Author(s):  
A. Espinel-Ingroff ◽  
A. L. Colombo ◽  
S. Cordoba ◽  
P. J. Dufresne ◽  
J. Fuller ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Fusarium Species ◽  
Gene Mutations ◽  
The United States ◽  
Wild Type ◽  
Content Type ◽  
Microdilution Method ◽  
Broth Microdilution Method ◽  
The Relationship ◽  
Dna Pcr

ABSTRACTThe CLSI epidemiological cutoff values (ECVs) of antifungal agents are available for variousCandidaspp.,Aspergillusspp., and the Mucorales. However, those categorical endpoints have not been established forFusariumspp., mostly due to the difficulties associated with collecting sufficient CLSI MICs for clinical isolates identified according to the currently recommended molecular DNA-PCR-based identification methodologies. CLSI MIC distributions were established for 53Fusarium dimerumspecies complex (SC), 10F. fujikuroi, 82F. proliferatum, 20F. incarnatum-F. equisetiSC, 226F. oxysporumSC, 608F. solaniSC, and 151F. verticillioidesisolates originating in 17 laboratories (in Argentina, Australia, Brazil, Canada, Europe, Mexico, and the United States). According to the CLSI guidelines for ECV setting, ECVs encompassing ≥97.5% of pooled statistically modeled MIC distributions were as follows: for amphotericin B, 4 μg/ml (F. verticillioides) and 8 μg/ml (F. oxysporumSC andF. solaniSC); for posaconazole, 2 μg/ml (F. verticillioides), 8 μg/ml (F. oxysporumSC), and 32 μg/ml (F. solaniSC); for voriconazole, 4 μg/ml (F. verticillioides), 16 μg/ml (F. oxysporumSC), and 32 μg/ml (F. solaniSC); and for itraconazole, 32 μg/ml (F. oxysporumSC andF. solaniSC). Insufficient data precluded ECV definition for the other species. Although these ECVs could aid in detecting non-wild-type isolates with reduced susceptibility to the agents evaluated, the relationship between molecular mechanisms of resistance (gene mutations) and MICs still needs to be investigated forFusariumspp.

scholarly journals Toxin Synthesis by Clostridium difficile Is Regulated through Quorum Signaling

mBio ◽  
2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Charles Darkoh ◽  
Herbert L. DuPont ◽  
Steven J. Norris ◽  
Heidi B. Kaplan
Keyword(s):  
Clostridium Difficile ◽  
Gut Microbiota ◽  
Antibiotic Therapy ◽  
Molecular Mechanisms ◽  
Public Health Problem ◽  
The United States ◽  
Multidrug Resistant ◽  
Accessory Gene ◽  
Content Type ◽  
Accessory Gene Regulator

ABSTRACTClostridium difficileinfection (CDI) is dramatically increasing as a cause of antibiotic- and hospital-associated diarrhea worldwide. C. difficile, a multidrug-resistant pathogen, flourishes in the colon after the gut microbiota has been altered by antibiotic therapy. Consequently, it produces toxins A and B that directly cause disease. Despite the enormous public health problem posed by this pathogen, the molecular mechanisms that regulate production of the toxins, which are directly responsible for disease, remained largely unknown until now. Here, we show that C. difficile toxin synthesis is regulated by an accessory gene regulator quorum-signaling system, which is mediated through a small (<1,000-Da) thiolactone that can be detected directly in stools of CDI patients. These findings provide direct evidence of the mechanism of regulation of C. difficile toxin synthesis and offer exciting new avenues both for rapid detection of C. difficile infection and development of quorum-signaling-based non-antibiotic therapies to combat this life-threatening emerging pathogen.IMPORTANCEClostridium difficileinfection (CDI) is the most common definable cause of hospital-acquired and antibiotic-associated diarrhea in the United States, with the total cost of treatment estimated between 1 and 4.8 billion U.S. dollars annually. C. difficile, a Gram-positive, spore-forming anaerobe, flourishes in the colon after the gut microbiota has been altered by antibiotic therapy. As a result, there is an urgent need for non-antibiotic CDI treatments that preserve the colonic microbiota. C. difficile produces toxins A and B, which are directly responsible for disease. Here, we report that C. difficile regulates its toxin synthesis by quorum signaling, in which a novel signaling peptide activates transcription of the disease-causing toxin genes. This finding provides new therapeutic targets to be harnessed for novel nonantibiotic therapy for C. difficile infections.

scholarly journals Dissemination of the Klebsiella pneumoniae Carbapenemase in the Health Care Settings: Tracking the Trails of an Elusive Offender

mBio ◽  
2011 ◽  
Vol 2 (6) ◽  
Author(s):  
Amos Adler ◽  
Yehuda Carmeli
Keyword(s):  
Klebsiella Pneumoniae ◽  
Resistance Genes ◽  
Molecular Mechanisms ◽  
Antibiotic Resistance Genes ◽  
The United States ◽  
Content Type ◽  
Modes Of Transmission ◽  
Klebsiella Pneumoniae Carbapenemase ◽  
Clone Sequence ◽  
Clonal Spread

ABSTRACT Transmission of antibiotic resistance genes may be mediated by a variety of molecular mechanisms, from mobility of small genetic elements to clonal spread. Since 1997, the carbapenem-hydrolyzing enzyme Klebsiella pneumoniae carbapenemase (KPC) has spread in the United States and across the world, mainly via a single K. pneumoniae clone, sequence type 258. By tracking the trail of dissemination of the bla KPC gene inside their institution, Mathers et al. (mBio 2:e00204–11, 2011) have shown evidence of the ability of this gene to spread by several modes, including plasmid transfer and clonal spread. The ever-evolving modes of transmission of resistance genes challenge our ability to detect, track, and eventually control the spread of what has become a major threat to hospitalized patients worldwide.

scholarly journals Microfluidic Chip-Based Detection and Intraspecies Strain Discrimination of Salmonella Serovars Derived from Whole Blood of Septic Mice

2013 ◽  
Vol 79 (7) ◽  
pp. 2302-2311 ◽  
Author(s):  
Adriana S. Patterson ◽  
Douglas M. Heithoff ◽  
Brian S. Ferguson ◽  
H. Tom Soh ◽  
Michael J. Mahan ◽  
...  
Keyword(s):  
Public Health ◽  
Microfluidic Chip ◽  
Whole Blood ◽  
Point Of Care ◽  
The United States ◽  
Isothermal Amplification ◽  
Microbial Pathogens ◽  
Content Type ◽  
Amplification Method ◽  
Single Temperature

ABSTRACTSalmonellais a zoonotic pathogen that poses a considerable public health and economic burden in the United States and worldwide. Resultant human diseases range from enterocolitis to bacteremia to sepsis and are acutely dependent on the particular serovar ofSalmonella entericasubsp.enterica, which comprises over 99% of human-pathogenicS. entericaisolates. Point-of-care methods for detection and strain discrimination ofSalmonellaserovars would thus have considerable benefit to medical, veterinary, and field applications that safeguard public health and reduce industry-associated losses. Here we describe a single, disposable microfluidic chip that supports isothermal amplification and sequence-specific detection and discrimination ofSalmonellaserovars derived from whole blood of septic mice. The integrated microfluidic electrochemical DNA (IMED) chip consists of an amplification chamber that supports loop-mediated isothermal amplification (LAMP), a rapid, single-temperature amplification method as an alternative to PCR that offers advantages in terms of sensitivity, reaction speed, and amplicon yield. The amplification chamber is connected via a microchannel to a detection chamber containing a reagentless, multiplexed (here biplex) sensing array for sequence-specific electrochemical DNA (E-DNA) detection of the LAMP products. Validation of the IMED device was assessed by the detection and discrimination ofS. entericasubsp.entericaserovars Typhimurium and Choleraesuis, the causative agents of enterocolitis and sepsis in humans, respectively. IMED chips conferred rapid (under 2 h) detection and discrimination of these strains at clinically relevant levels (<1,000 CFU/ml) from whole, unprocessed blood collected from septic animals. The IMED-based chip assay shows considerable promise as a rapid, inexpensive, and portable point-of-care diagnostic platform for the detection and strain-specific discrimination of microbial pathogens.

scholarly journals The Periodontal Pathogen Porphyromonas gingivalis Induces Expression of Transposases and Cell Death of Streptococcus mitis in a Biofilm Model

2014 ◽  
Vol 82 (8) ◽  
pp. 3374-3382 ◽  
Author(s):  
Ana E. Duran-Pinedo ◽  
Vinesha D. Baker ◽  
Jorge Frias-Lopez
Keyword(s):  
Cell Death ◽  
Microbial Community ◽  
Porphyromonas Gingivalis ◽  
Bacterial Species ◽  
The United States ◽  
Oral Microbiome ◽  
Microbial Pathogens ◽  
Periodontal Pathogen ◽  
Streptococcus Mitis ◽  
Content Type

ABSTRACTOral microbial communities are extremely complex biofilms with high numbers of bacterial species interacting with each other (and the host) to maintain homeostasis of the system. Disturbance in the oral microbiome homeostasis can lead to either caries or periodontitis, two of the most common human diseases. Periodontitis is a polymicrobial disease caused by the coordinated action of a complex microbial community, which results in inflammation of tissues that support the teeth. It is the most common cause of tooth loss among adults in the United States, and recent studies have suggested that it may increase the risk for systemic conditions such as cardiovascular diseases. In a recent series of papers, Hajishengallis and coworkers proposed the idea of the “keystone-pathogen” where low-abundance microbial pathogens (Porphyromonas gingivalis) can orchestrate inflammatory disease by turning a benign microbial community into a dysbiotic one. The exact mechanisms by which these pathogens reorganize the healthy oral microbiome are still unknown. In the present manuscript, we present results demonstrating thatP. gingivalisinducesS. mitisdeath and DNA fragmentation in anin vitrobiofilm system. Moreover, we report here the induction of expression of multiple transposases in aStreptococcus mitisbiofilm when the periodontopathogenP. gingivalisis present. Based on these results, we hypothesize thatP. gingivalisinducesS. mitiscell death by an unknown mechanism, shaping the oral microbiome to its advantage.

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