Relationship of Virulence Factor Expression to Evolved Virulence in Mouse-Passaged Cryptococcus neoformans Lines

ABSTRACT Serial passage of Cryptococcus neoformans in mice increases virulence relative to the nonpassaged line. Postpassaged lines showed no difference in the expression of most known virulence factors, with the exception that the more virulent lines had smaller capsules in vitro. These data imply that other mechanisms of virulence remain to be discovered.

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A Salmonella Typhi RNA thermosensor regulates virulence factors and innate immune evasion in response to host temperature

PLoS Pathogens ◽

10.1371/journal.ppat.1009345 ◽

2021 ◽

Vol 17 (3) ◽

pp. e1009345

Author(s):

Susan M. Brewer ◽

Christian Twittenhoff ◽

Jens Kortmann ◽

Sky W. Brubaker ◽

Jared Honeycutt ◽

...

Keyword(s):

Secondary Structure ◽

Virulence Factors ◽

Virulence Factor ◽

Structure Prediction ◽

Secondary Structure Prediction ◽

Bacterial Pathogens ◽

Innate Immune ◽

New Host ◽

Factor Expression

Sensing and responding to environmental signals is critical for bacterial pathogens to successfully infect and persist within hosts. Many bacterial pathogens sense temperature as an indication they have entered a new host and must alter their virulence factor expression to evade immune detection. Using secondary structure prediction, we identified an RNA thermosensor (RNAT) in the 5’ untranslated region (UTR) of tviA encoded by the typhoid fever-causing bacterium Salmonella enterica serovar Typhi (S. Typhi). Importantly, tviA is a transcriptional regulator of the critical virulence factors Vi capsule, flagellin, and type III secretion system-1 expression. By introducing point mutations to alter the mRNA secondary structure, we demonstrate that the 5’ UTR of tviA contains a functional RNAT using in vitro expression, structure probing, and ribosome binding methods. Mutational inhibition of the RNAT in S. Typhi causes aberrant virulence factor expression, leading to enhanced innate immune responses during infection. In conclusion, we show that S. Typhi regulates virulence factor expression through an RNAT in the 5’ UTR of tviA. Our findings demonstrate that limiting inflammation through RNAT-dependent regulation in response to host body temperature is important for S. Typhi’s “stealthy” pathogenesis.

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Serial Passage of Cryptococcus neoformans in Galleria mellonella Results in Increased Capsule and Intracellular Replication in Hemocytes, but Not Increased Resistance to Hydrogen Peroxide

Pathogens ◽

10.3390/pathogens9090732 ◽

2020 ◽

Vol 9 (9) ◽

pp. 732 ◽

Cited By ~ 1

Author(s):

Muhammad Fariz Ali ◽

Stephen M. Tansie ◽

John R. Shahan ◽

Rebecca L. Seipelt-Thiemann ◽

Erin E. McClelland

Keyword(s):

Hydrogen Peroxide ◽

Cryptococcus Neoformans ◽

Galleria Mellonella ◽

Ex Vivo ◽

Histopathological Examination ◽

Serial Passage ◽

Infection Model ◽

Hydrogen Peroxide Production ◽

Species Response

To gain insight into how pathogens adapt to new hosts, Cryptococcus neoformans (H99W) was serially passaged in Galleria mellonella. The phenotypic characteristics of the passaged strain (P15) and H99W were evaluated. P15 grew faster in hemolymph than H99W, in vitro and in vivo, suggesting that adaptation had occurred. However, P15 was more susceptible to hydrogen peroxide in vitro, killed fewer mouse macrophages, and had less fungal burden in human ex vivo macrophages than H99W. Analysis of gene expression changes during Galleria infection showed only a few different genes involved in the reactive oxygen species response. As P15 sheds more GXM than H99W, P15 may have adapted by downregulating hemocyte hydrogen peroxide production, possibly through increased capsular glucuronoxylomannan (GXM) shedding. Hemocytes infected with P15 produced less hydrogen peroxide, and hydrogen peroxide production in response to GXM-shedding mutants was correlated with shed GXM. Histopathological examination of infected larvae showed increased numbers and sizes of immune nodules for P15 compared to H99W, suggesting an enhanced, but functionally defective, response to P15. These results could explain why this infection model does not always correlate with murine models. Overall, C. neoformans’ serial passage in G. mellonella resulted in a better understanding of how this yeast evolves under selection.

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Sequence Analysis of the Staphylococcus aureus srrAB Loci Reveals that Truncation of srrA Affects Growth and Virulence Factor Expression

Journal of Bacteriology ◽

10.1128/jb.00547-07 ◽

2007 ◽

Vol 189 (20) ◽

pp. 7515-7519 ◽

Cited By ~ 11

Author(s):

Alexa A. Pragman ◽

Lisa Herron-Olson ◽

Laura C. Case ◽

Sara M. Vetter ◽

Evan E. Henke ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Phylogenetic Analysis ◽

Sequence Analysis ◽

Virulence Factors ◽

Virulence Factor ◽

Nucleotide Polymorphisms ◽

Low Oxygen ◽

Factor Expression ◽

Oxygen Conditions

ABSTRACT The SrrAB system regulates metabolism and virulence factors in Staphylococcus aureus. We sequenced the srrAB loci of 21 isolates and performed a phylogenetic analysis. Vaginal and bovine isolates clustered together, while skin isolates were genetically diverse. Few nucleotide polymorphisms were observed, and most were synonymous. Two strains (N2 and N19) with N-terminal truncations in SrrA displayed defects in growth and abnormally upregulated virulence factor expression under low-oxygen conditions.

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Cryptococcus neoformans var. grubii isolates recovered from persons with AIDS demonstrate a wide range of virulence during murine meningoencephalitis that correlates with the expression of certain virulence factors

Microbiology ◽

10.1099/mic.0.28798-0 ◽

2006 ◽

Vol 152 (8) ◽

pp. 2247-2255 ◽

Cited By ~ 26

Author(s):

Cornelius J. Clancy ◽

M. Hong Nguyen ◽

Ruth Alandoerffer ◽

Shaoji Cheng ◽

Kenneth Iczkowski ◽

...

Keyword(s):

Cryptococcus Neoformans ◽

Virulence Factors ◽

Survival Rates ◽

Dominant Factor ◽

Aids Patients ◽

Wide Range ◽

Survival Studies ◽

Capsule Size

Cryptococcus neoformans is a common cause of meningoencephalitis among AIDS patients. Several C. neoformans virulence factors have been identified, but the relative importance of particular factors is unknown. This study examined the corrrelation of the virulence of 18 C. neoformans var. grubii isolates from AIDS patients with the expression of several well-described virulence factors. The LD50 at 15 days after intracranial inoculation of ICR mice was <100 c.f.u. for 22 % of isolates, 100–1000 for 28 %, 1000–10 000 for 11 % and >20 000 for 39 %. Higher cryptococcal concentrations in brains were noted for isolates with lower LD50 (P=0.002). In survival studies, no immunocompetent BALB/c mice (nu/−) infected with 3×LD50 of three virulent isolates (LD50=62, 99, 1280) survived beyond 23 days, whereas 100 %, 90 % and 90 % of mice infected with 20 000 c.f.u. of three hypovirulent isolates (LD50>20 000) survived for 60 days (P<0.0001). Even among BALB/c nude (nu/nu) mice, survival rates over 60 days were 100 %, 70 % and 50 %, respectively, for the hypovirulent isolates. Growth rate at 37 °C and capsule size within brains correlated with LD50 by univariate (P=0.0001 and 0.028, respectively) and multivariate (P=0.017 and 0.016, respectively) analyses. There was no correlation between LD50 and capsule size in vitro, phospholipase activity, melanin formation, proteinase activity and fluconazole MIC. In conclusion, AIDS patients are susceptible to infection by C. neoformans isolates of wide-ranging virulence, including isolates that are markedly hypovirulent. The virulence of a given isolate reflects a composite of factors rather than the contribution of a dominant factor. Growth at 37 °C and capsule size in vivo make particularly important contributions.

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Cryptococcal Phospholipase B1 Is Required for Intracellular Proliferation and Control of Titan Cell Morphology during Macrophage Infection

Infection and Immunity ◽

10.1128/iai.03104-14 ◽

2015 ◽

Vol 83 (4) ◽

pp. 1296-1304 ◽

Cited By ~ 32

Author(s):

Robert J. Evans ◽

Zhongming Li ◽

William S. Hughes ◽

Julianne T. Djordjevic ◽

Kirsten Nielsen ◽

...

Keyword(s):

Cryptococcus Neoformans ◽

Virulence Factors ◽

Fold Increase ◽

Macrophage Infection ◽

Content Type ◽

The Central Nervous System ◽

Opportunistic Fungal Pathogen ◽

And Control

Cryptococcus neoformansis an opportunistic fungal pathogen and a leading cause of fungal-infection-related fatalities, especially in immunocompromised hosts. Several virulence factors are known to play a major role in the pathogenesis of cryptococcal infections, including the enzyme phospholipase B1 (Plb1). Compared to other well-studiedCryptococcus neoformansvirulence factors such as the polysaccharide capsule and melanin production, very little is known about the contribution of Plb1 to cryptococcal virulence. Phospholipase B1 is a phospholipid-modifying enzyme that has been implicated in multiple stages of cryptococcal pathogenesis, including initiation and persistence of pulmonary infection and dissemination to the central nervous system, but the underlying reason for these phenotypes remains unknown. Here we demonstrate that a Δplb1knockout strain ofC. neoformanshas a profound defect in intracellular growth within host macrophages. This defect is due to a combination of a 50% decrease in proliferation and a 2-fold increase in cryptococcal killing within the phagosome. In addition, we show for the first time that the Δplb1strain undergoes a morphological change duringin vitroandin vivointracellular infection, resulting in a subpopulation of very large titan cells, which may arise as a result of the attenuated mutant's inability to cope within the macrophage.

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Characterisation of Clostridium perfringens isolated from chickens in Vietnam

Veterinární Medicína ◽

10.17221/209/2020-vetmed ◽

2021 ◽

Vol 66 (No. 10) ◽

pp. 431-439

Author(s):

TN Thi ◽

H Vu-Khac ◽

TN Duc

Keyword(s):

Multiplex Pcr ◽

Virulence Factors ◽

Clostridium Perfringens ◽

Virulence Factor ◽

Necrotic Enteritis ◽

Sds Page ◽

Major Player ◽

Positive Isolate

The objective of this study was isolating and characterising Clostridium perfringens from chickens in Vietnam and identifying virulence factors involved with enteritis. Five hundred thirty-one faecal and sixty-eight intestinal samples were collected from healthy and diseased chickens for the C. perfringens isolation. The presence of virulence factors was determined by multiplex PCR. The netB gene of the selected isolates was sequenced and checked for its expression by SDS-PAGE. Two hundred seventy-two C. perfringens isolates were collected. All of them were shown to be positive for the cpa gene. The netB gene was detected in 26.56% of the C. perfringens isolates from the healthy chickens, while 43.45% of the isolates from the faeces and 45% of the isolates from the intestinal samples were positive for this gene in the diseased birds. All eight isolates positive to netB from the diseased chickens showed 100% identity in the netB sequence and produced the NetB toxin in vitro, whereas only two out of eight healthy chicken-derived isolates produced this toxin. Nine out of ten chickens experimentally infected with the C. perfringens netB-positive isolate showed typical signs of enteritis. The cpa gene was the most prevalent virulence factor identified in the bacteria C. perfringens, but the netB gene could be a major player responsible for necrotic enteritis progression in chickens.

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An Intracellular Peptidyl-Prolyl cis/trans Isomerase Is Required for Folding and Activity of the Staphylococcus aureus Secreted Virulence Factor Nuclease

Journal of Bacteriology ◽

10.1128/jb.00453-16 ◽

2016 ◽

Vol 199 (1) ◽

Cited By ~ 16

Author(s):

Richard E. Wiemels ◽

Stephanie M. Cech ◽

Nikki M. Meyer ◽

Caleb A. Burke ◽

Andy Weiss ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Virulence Factors ◽

Virulence Factor ◽

Active Form ◽

Ppiase Activity ◽

Content Type ◽

Associated Proteins

ABSTRACT Staphylococcus aureus is an important human pathogen that relies on a large repertoire of secreted and cell wall-associated proteins for pathogenesis. Consequently, the ability of the organism to cause disease is absolutely dependent on its ability to synthesize and successfully secrete these proteins. In this study, we investigate the role of peptidyl-prolyl cis/trans isomerases (PPIases) on the activity of the S. aureus secreted virulence factor nuclease (Nuc). We identify a staphylococcal cyclophilin-type PPIase (PpiB) that is required for optimal activity of Nuc. Disruption of ppiB results in decreased nuclease activity in culture supernatants; however, the levels of Nuc protein are not altered, suggesting that the decrease in activity results from misfolding of Nuc in the absence of PpiB. We go on to demonstrate that PpiB exhibits PPIase activity in vitro, is localized to the bacterial cytosol, and directly interacts with Nuc in vitro to accelerate the rate of Nuc refolding. Finally, we demonstrate an additional role for PpiB in S. aureus hemolysis and demonstrate that the S. aureus parvulin-type PPIase PrsA also plays a role in the activity of secreted virulence factors. The deletion of prsA leads to a decrease in secreted protease and phospholipase activity, similar to that observed in other Gram-positive pathogens. Together, these results demonstrate, for the first time to our knowledge, that PPIases play an important role in the secretion of virulence factors in S. aureus. IMPORTANCE Staphylococcus aureus is a highly dangerous bacterial pathogen capable of causing a variety of infections throughout the human body. The ability of S. aureus to cause disease is largely due to an extensive repertoire of secreted and cell wall-associated proteins, including adhesins, toxins, exoenzymes, and superantigens. These virulence factors, once produced, are typically transported across the cell membrane by the secretory (Sec) system in a denatured state. Consequently, once outside the cell, they must refold into their active form. This step often requires the assistance of bacterial folding proteins, such as PPIases. In this work, we investigate the role of PPIases in S. aureus and uncover a cyclophilin-type enzyme that assists in the folding/refolding of staphylococcal nuclease.

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Disarming Fungal Pathogens:Bacillus safensisInhibits Virulence Factor Production and Biofilm Formation byCryptococcus neoformansandCandida albicans

mBio ◽

10.1128/mbio.01537-17 ◽

2017 ◽

Vol 8 (5) ◽

Cited By ~ 24

Author(s):

François L. Mayer ◽

James W. Kronstad

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Cryptococcus Neoformans ◽

Virulence Factors ◽

Virulence Factor ◽

Biofilm Formation ◽

Fungal Pathogens ◽

Soil Bacterium ◽

Fungal Cell ◽

Virulence Factor Production

ABSTRACTBacteria interact with each other in nature and often compete for limited nutrient and space resources. However, it is largely unknown whether and how bacteria also interact with human fungal pathogens naturally found in the environment. Here, we identified a soil bacterium,Bacillus safensis, which potently blocked several keyCryptococcus neoformansvirulence factors, including formation of the antioxidant pigment melanin and production of the antiphagocytic polysaccharide capsule. The bacterium also inhibitedde novocryptococcal biofilm formation but had only modest inhibitory effects on already formed biofilms or planktonic cell growth. The inhibition of fungal melanization was dependent on direct cell contact and live bacteria.B. safensisalso had anti-virulence factor activity against another major human-associated fungal pathogen,Candida albicans. Specifically, dual-species interaction studies revealed that the bacterium strongly inhibitedC. albicansfilamentation and biofilm formation. In particular,B. safensisphysically attached to and degraded candidal filaments. Through genetic and phenotypic analyses, we demonstrated that bacterial chitinase activity against fungal cell wall chitin is a factor contributing to the antipathogen effect ofB. safensis.IMPORTANCEPathogenic fungi are estimated to contribute to as many human deaths as tuberculosis or malaria. Two of the most common fungal pathogens,Cryptococcus neoformansandCandida albicans, account for up to 1.4 million infections per year with very high mortality rates. Few antifungal drugs are available for treatment, and development of novel therapies is complicated by the need for pathogen-specific targets. Therefore, there is an urgent need to identify novel drug targets and new drugs. Pathogens use virulence factors during infection, and it has recently been proposed that targeting these factors instead of the pathogen itself may represent a new approach to develop antimicrobials. Here, we identified a soil bacterium that specifically blocked virulence factor production and biofilm formation byC. neoformansandC. albicans. We demonstrate that the bacterial antipathogen mechanism is based in part on targeting the fungal cell wall, a structure not found in human cells.

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Environmental Signals Modulate ToxT-Dependent Virulence Factor Expression in Vibrio cholerae

Journal of Bacteriology ◽

10.1128/jb.181.5.1508-1514.1999 ◽

1999 ◽

Vol 181 (5) ◽

pp. 1508-1514 ◽

Cited By ~ 117

Author(s):

Darren A. Schuhmacher ◽

Karl E. Klose

Keyword(s):

Vibrio Cholerae ◽

Virulence Factors ◽

Virulence Factor ◽

Transcriptional Activation ◽

Regulatory Protein ◽

Inducible Promoter ◽

Negative Regulation ◽

Transcriptional Level ◽

Environmental Signals ◽

Factor Expression

ABSTRACT The regulatory protein ToxT directly activates the transcription of virulence factors in Vibrio cholerae, including cholera toxin (CT) and the toxin-coregulated pilus (TCP). Specific environmental signals stimulate virulence factor expression by inducing the transcription of toxT. We demonstrate that transcriptional activation by the ToxT protein is also modulated by environmental signals. ToxT expressed from an inducible promoter activated high-level expression of CT and TCP in V. cholerae at 30°C, but expression of CT and TCP was significantly decreased or abolished by the addition of 0.4% bile to the medium and/or an increase of the temperature to 37°C. Also, expression of six ToxT-dependent TnphoA fusions was modulated by temperature and bile. Measurement of ToxT-dependent transcription of genes encoding CT and TCP by ctxAp- andtcpAp-luciferase fusions confirmed that negative regulation by 37°C or bile occurs at the transcriptional level in V. cholerae. Interestingly, ToxT-dependent transcription of these same promoters in Salmonella typhimurium was relatively insensitive to regulation by temperature or bile. These data are consistent with ToxT transcriptional activity being modulated by environmental signals in V. cholerae and demonstrate an additional level of complexity governing the expression of virulence factors in this pathogen. We propose that negative regulation of ToxT-dependent transcription by environmental signals prevents the incorrect temporal and spatial expression of virulence factors during cholera pathogenesis.

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Characterization of the Role of the ToxR-Modulated Outer Membrane Porins OmpU and OmpT in Vibrio cholerae Virulence

Journal of Bacteriology ◽

10.1128/jb.183.12.3652-3662.2001 ◽

2001 ◽

Vol 183 (12) ◽

pp. 3652-3662 ◽

Cited By ~ 66

Author(s):

Daniele Provenzano ◽

Crystal M. Lauriano ◽

Karl E. Klose

Keyword(s):

Virulence Factor ◽

Large Deletion ◽

Wild Type ◽

Coding Sequence ◽

Rich Media ◽

Outer Membrane Porins ◽

Factor Expression

ABSTRACT ToxR, the transmembrane regulatory protein required for expression of virulence factors in the human diarrheal pathogen Vibrio cholerae, directly activates and represses the transcription of two outer membrane porins, OmpU and OmpT, respectively. In an attempt to dissect the role of the OmpU and OmpT porins in viability and virulence factor expression, in-frame chromosomal deletions were constructed in the coding sequences of ompU andompT of V. cholerae. Two separate deletions were introduced into ompU; the first (small) deletion, ΔompU1, removed the coding sequence for 84 internal amino acids (aa), while the second (large) deletion, ΔompU2, removed the coding sequence for the entire amino-terminal 274 aa. The ΔompU1 strain had a growth defect that could not be complemented by episomal expression of full-length ompU. In contrast, a strain with ΔompU2 displayed wild-type growth kinetics in rich media, suggesting that this is the true phenotype of a strain lacking OmpU and that the truncated OmpU protein, rather than the absence of OmpU, may be the cause for the ΔompU1phenotype. A large deletion removing the coding sequence for the entire N-terminal 273 aa of OmpT (ΔompT) was also constructed in wild-type as well as ΔtoxR and ΔompU2strains, and these strains displayed wild-type growth kinetics in rich media. However, the ΔompU2 strain was deficient for growth in deoxycholate compared to wild-type, ΔompT, and ΔompU2 ΔompT strains, reinforcing a positive role for the OmpU porin and a negative role for the OmpT porin in V. cholerae resistance to anionic detergents. The ΔompU2, ΔompT, and ΔompU2ΔompT strains exhibited wild-type levels of in vitro virulence factor expression and resistance to polymyxin B and serum and in vivo colonization levels similar to a wild-type strain in the infant mouse intestine. Our results demonstrate that (i) OmpU and OmpT are not essential proteins, as was previously thought; (ii) these porins contribute to V. cholerae resistance to anionic detergents; and (iii) OmpU and OmpT are not essential for virulence factor expression in vitro or intestinal colonization in vivo.

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