scholarly journals Null Mutations of Group A Streptococcus Orphan Kinase RocA: Selection in Mouse Infection and Comparison with CovS Mutations in Alteration of In Vitro and In Vivo Protease SpeB Expression and Virulence

2016 ◽  
Vol 85 (1) ◽  
Author(s):  
Wenchao Feng ◽  
Dylan Minor ◽  
Mengyao Liu ◽  
Jinquan Li ◽  
Suzanne L. Ishaq ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Phase ◽  
Virulence Genes ◽  
Expression Patterns ◽  
Mouse Skin ◽  
Exponential Growth Phase ◽  
Wild Type ◽  
Subcutaneous Infection ◽  
Group A

ABSTRACT Group A Streptococcus (GAS) acquires mutations of the virulence regulator CovRS in human and mouse infections, and these mutations result in the upregulation of virulence genes and the downregulation of the protease SpeB. To identify in vivo mutants with novel phenotypes, GAS isolates from infected mice were screened by enzymatic assays for SpeB and the platelet-activating factor acetylhydrolase Sse, and a new type of variant that had enhanced Sse expression and normal levels of SpeB production was identified (the variants had a phenotype referred to as enhanced Sse activity [SseA+] and normal SpeB activity [SpeBA+]). SseA+ SpeBA+ variants had transcript levels of CovRS-controlled virulence genes comparable to those of a covS mutant but had no covRS mutations. Genome resequencing of an SseA+ SpeBA+ isolate identified a C605A nonsense mutation in orphan kinase gene rocA, and 6 other SseA+ SpeBA+ isolates also had nonsense mutations or small indels in rocA. RocA and CovS mutants had similar levels of enhancement of the expression of CovRS-controlled virulence genes at the exponential growth phase; however, mutations of RocA but not mutations of CovS did not result in the downregulation of speB transcription at stationary growth phase or in subcutaneous infection of mice. GAS with RocA and CovS mutations caused greater enhancement of the expression of hasA than spyCEP in mouse skin infection than wild-type GAS did. RocA mutants ranked between wild-type GAS and CovS mutants in skin invasion, inhibition of neutrophil recruitment, and virulence in subcutaneous infection of mice. Thus, GAS RocA mutants can be selected in subcutaneous infections in mice and exhibit gene expression patterns and virulences distinct from those of CovS mutants. The findings provide novel information for understanding GAS fitness mutations in vivo, virulence gene regulation, in vivo gene expression, and virulence.

scholarly journals Active and Passive Immunizations with the Streptococcal Esterase Sse Protect Mice against Subcutaneous Infection with Group A Streptococci

2007 ◽  
Vol 75 (7) ◽  
pp. 3651-3657 ◽  
Author(s):  
Mengyao Liu ◽  
Hui Zhu ◽  
Jinlian Zhang ◽  
Benfang Lei
Keyword(s):  
Protective Antigen ◽  
Passive Immunization ◽  
Mouse Skin ◽  
Streptococcal Pharyngitis ◽  
Active Immunization ◽  
Subcutaneous Infection ◽  
Group A ◽  
Esterase Gene

ABSTRACT The human pathogen group A Streptococcus (GAS) produces many secreted proteins that play important roles in GAS pathogenesis, including hydrolases that degrade proteins and nucleic acids. This study targets another kind of hydrolase, carboxylic esterase, with the objectives of identifying GAS esterase and determining whether it is a protective antigen. The putative esterase gene SPy1718 was cloned, and the recombinant protein (Sse) was prepared. Sse was detected in GAS culture supernatant, and patients with streptococcal pharyngitis seroconverted to Sse, indicating that Sse was produced in vivo and in vitro. Sse hydrolyzes p-nitrophenyl butyrate, and the residue 178Ser is critical for this esterase activity. There are two Sse variant complexes according to the available genome databases, consistent with the previous finding of two antigenic Sse variants. Complex I includes serotypes M1, M2, M3, M5, M6, M12, and M18, whereas M4, M28, and M49 belong to complex II. Sse variants share >98% identity in amino acid sequence within each complex but have about 37% variation between the two groups. Active immunization with M1 Sse significantly protects mice against lethal subcutaneous infection with virulent M1 and M3 strains and inhibits GAS invasion of mouse skin tissue. Passive immunization with anti-Sse antiserum also significantly protects mice against subcutaneous GAS infection, indicating that the protection is mediated by Sse-specific antibodies. The results suggest that Sse plays an important role in tissue invasion and is an antigen protective in subcutaneous infection against GAS strains of more than one serotype.

scholarly journals Regulation of SpeB in Streptococcus pyogenes by pH and NaCl: a Model for In Vivo Gene Expression

2006 ◽  
Vol 188 (2) ◽  
pp. 399-408 ◽  
Author(s):  
Jennifer A. Loughman ◽  
Michael Caparon
Keyword(s):  
Gene Expression ◽  
Streptococcus Pyogenes ◽  
Growth Phase ◽  
Soft Tissues ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Transcriptional Response ◽  
Culture Conditions

ABSTRACT For a pathogen such as Streptococcus pyogenes, ecological success is determined by its ability to sense the environment and mount an appropriate adaptive transcriptional response. Thus, determining conditions for analyses of gene expression in vitro that are representative of the in vivo environment is critical for understanding the contributions of transcriptional response pathways to pathogenesis. In this study, we determined that the gene encoding the SpeB cysteine protease is up-regulated over the course of infection in a murine soft-tissue model. Conditions were identified, including growth phase, acidic pH, and an NaCl concentration of <0.1 M, that were required for expression of speB in vitro. Analysis of global expression profiles in response to these conditions in vitro identified a set of coregulated genes whose expression patterns showed a significant correlation with that of speB when examined during infection of murine soft tissues. This analysis revealed that a culture medium that promotes high levels of SpeB expression in vitro produced an expression profile that showed significant correlation to the profile observed in vivo. Taken together, these studies establish culture conditions that mimic in vivo expression patterns; that growth phase, pH, and NaCl may mimic relevant cues sensed by S. pyogenes during infection; and that identification of other environmental cues that alter expression of speB in vitro may provide insight into the signals that direct global patterns of gene expression in vivo.

scholarly journals CovS Simultaneously Activates and Inhibits the CovR-Mediated Repression of Distinct Subsets of Group A Streptococcus Virulence Factor-Encoding Genes

2009 ◽  
Vol 77 (8) ◽  
pp. 3141-3149 ◽  
Author(s):  
Jeanette Treviño ◽  
Nataly Perez ◽  
Esmeralda Ramirez-Peña ◽  
Zhuyun Liu ◽  
Samuel A. Shelburne ◽  
...  
Keyword(s):  
Gene Expression ◽  
Molecular Mechanisms ◽  
Regulatory Function ◽  
Upper Respiratory Tract ◽  
Wild Type ◽  
In Vivo Models ◽  
Mutant Strains ◽  
Invasive Infections ◽  
Group A

ABSTRACT To colonize and cause disease at distinct anatomical sites, bacterial pathogens must tailor gene expression in a microenvironment-specific manner. The molecular mechanisms that control the ability of the human bacterial pathogen group A Streptococcus (GAS) to transition between infection sites have yet to be fully elucidated. A key regulator of GAS virulence gene expression is the CovR-CovS two-component regulatory system (also known as CsrR-CsrS). covR and covS mutant strains arise spontaneously during invasive infections and, in in vivo models of infection, rapidly become dominant. Here, we compared wild-type GAS with covR, covS, and covRS isogenic mutant strains to investigate the heterogeneity in the types of natural mutations that occur in covR and covS and the phenotypic consequences of covR or covS mutation. We found that the response regulator CovR retains some regulatory function in the absence of CovS and that CovS modulates CovR to significantly enhance repression of one group of genes (e.g., the speA, hasA, and ska genes) while it reduces repression of a second group of genes (e.g., the speB, grab, and spd3 genes). We also found that different in vivo-induced covR mutations can lead to strikingly different transcriptomes. While covS mutant strains show increased virulence in several invasive models of infection, we determined that these mutants are significantly outcompeted by wild-type GAS during growth in human saliva, an ex vivo model of upper respiratory tract infection. We propose that CovS-mediated regulation of CovR activity plays an important role in the ability of GAS to cycle between pharyngeal and invasive infections.

scholarly journals Phosphorylation Events in the Multiple Gene Regulator of Group A Streptococcus Significantly Influence Global Gene Expression and Virulence

2015 ◽  
Vol 83 (6) ◽  
pp. 2382-2395 ◽  
Author(s):  
Misu Sanson ◽  
Nishanth Makthal ◽  
Maire Gavagan ◽  
Concepcion Cantu ◽  
Randall J. Olsen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Mutant Strain ◽  
Group A Streptococcus ◽  
Global Gene Expression ◽  
Sequencing Analysis ◽  
Wild Type ◽  
Content Type ◽  
Group A

Whole-genome sequencing analysis of ∼800 strains of group AStreptococcus(GAS) found that the gene encoding themultiple virulencegene regulator of GAS (mga) is highly polymorphic in serotype M59 strains but not in strains of other serotypes. To help understand the molecular mechanism of gene regulation by Mga and its contribution to GAS pathogenesis in serotype M59 GAS, we constructed an isogenicmgamutant strain. Transcriptome studies indicated a significant regulatory influence of Mga and altered metabolic capabilities conferred by Mga-regulated genes. We assessed the phosphorylation status of Mga in GAS cell lysates with Phos-tag gels. The results revealed that Mga is phosphorylated at histidinesin vivo. Using phosphomimetic and nonphosphomimetic substitutions at conserved phosphoenolpyruvate:carbohydrate phosphotransferase regulation domain (PRD) histidines of Mga, we demonstrated that phosphorylation-mimicking aspartate replacements at H207 and H273 of PRD-1 and at H327 of PRD-2 are inhibitory to Mga-dependent gene expression. Conversely, non-phosphorylation-mimicking alanine substitutions at H273 and H327 relieved inhibition, and the mutant strains exhibited a wild-type phenotype. The opposing regulatory profiles observed for phosphorylation- and non-phosphorylation-mimicking substitutions at H273 extended to global gene regulation by Mga. Consistent with these observations, the H273D mutant strain attenuated GAS virulence, whereas the H273A strain exhibited a wild-type virulence phenotype in a mouse model of necrotizing fasciitis. Together, our results demonstrate phosphoregulation of Mga and its direct link to virulence in M59 GAS strains. These data also lay a foundation toward understanding how naturally occurring gain-of-function variations inmga, such as H201R, may confer an advantage to the pathogen and contribute to M59 GAS pathogenesis.

Vibrio harveyi virulence gene expression in vitro and in vivo during infection in black tiger shrimp Penaeus monodon

2020 ◽  
Vol 139 ◽  
pp. 153-160
Author(s):  
S Peeralil ◽  
TC Joseph ◽  
V Murugadas ◽  
PG Akhilnath ◽  
VN Sreejith ◽  
...  
Keyword(s):  
Gene Expression ◽  
Virulence Genes ◽  
Vibrio Harveyi ◽  
Penaeus Monodon ◽  
Challenge Test ◽  
Virulence Gene ◽  
Economic Losses ◽  
Virulence Gene Expression

Luminescent Vibrio harveyi is common in sea and estuarine waters. It produces several virulence factors and negatively affects larval penaeid shrimp in hatcheries, resulting in severe economic losses to shrimp aquaculture. Although V. harveyi is an important pathogen of shrimp, its pathogenicity mechanisms have yet to be completely elucidated. In the present study, isolates of V. harveyi were isolated and characterized from diseased Penaeus monodon postlarvae from hatcheries in Kerala, India, from September to December 2016. All 23 tested isolates were positive for lipase, phospholipase, caseinase, gelatinase and chitinase activity, and 3 of the isolates (MFB32, MFB71 and MFB68) showed potential for significant biofilm formation. Based on the presence of virulence genes, the isolates of V. harveyi were grouped into 6 genotypes, predominated by vhpA+ flaB+ ser+ vhh1- luxR+ vopD- vcrD+ vscN-. One isolate from each genotype was randomly selected for in vivo virulence experiments, and the LD50 ranged from 1.7 ± 0.5 × 103 to 4.1 ± 0.1 × 105 CFU ml-1. The expression of genes during the infection in postlarvae was high in 2 of the isolates (MFB12 and MFB32), consistent with the result of the challenge test. However, in MFB19, even though all genes tested were present, their expression level was very low and likely contributed to its lack of virulence. Because of the significant variation in gene expression, the presence of virulence genes alone cannot be used as a marker for pathogenicity of V. harveyi.

scholarly journals Compendium of Methods to Uncover RNA-Protein Interactions In Vivo

2021 ◽  
Vol 4 (1) ◽  
pp. 22
Author(s):  
Mrinmoyee Majumder ◽  
Viswanathan Palanisamy
Keyword(s):  
Gene Expression ◽  
Protein Interactions ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Expression Patterns ◽  
Control Of Gene Expression ◽  
Regulatory Pathways ◽  
Advantages And Disadvantages ◽  
Protein Nucleic Acid

Control of gene expression is critical in shaping the pro-and eukaryotic organisms’ genotype and phenotype. The gene expression regulatory pathways solely rely on protein–protein and protein–nucleic acid interactions, which determine the fate of the nucleic acids. RNA–protein interactions play a significant role in co- and post-transcriptional regulation to control gene expression. RNA-binding proteins (RBPs) are a diverse group of macromolecules that bind to RNA and play an essential role in RNA biology by regulating pre-mRNA processing, maturation, nuclear transport, stability, and translation. Hence, the studies aimed at investigating RNA–protein interactions are essential to advance our knowledge in gene expression patterns associated with health and disease. Here we discuss the long-established and current technologies that are widely used to study RNA–protein interactions in vivo. We also present the advantages and disadvantages of each method discussed in the review.

scholarly journals Recent Progress in the Study of Peroxiredoxin in the Harmful Algal Bloom Species Chattonella marina

Antioxidants ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 162
Author(s):  
Yohei Shimasaki ◽  
Koki Mukai ◽  
Yuki Takai ◽  
Xuchun Qiu ◽  
Yuji Oshima
Keyword(s):  
Oxidative Stress ◽  
Growth Phase ◽  
Critical Role ◽  
High Irradiance ◽  
Growth Potential ◽  
Exponential Growth Phase ◽  
Wild Type ◽  
Chattonella Marina ◽  
Strong Light ◽  
Expression Strain

Peroxiredoxin (Prx) is a relatively recently discovered antioxidant enzyme family that scavenges peroxides and is known to be present in organisms from biological taxa ranging from bacteria to multicellular eukaryotes, including photosynthetic organisms. Although there have been many studies of the Prx family in higher plants, green algae, and cyanobacteria, few studies have concerned raphidophytes and dinoflagellates, which are among the eukaryotic algae that cause harmful algal blooms (HABs). In our proteomic study using 2-D electrophoresis, we found a highly expressed 2-Cys peroxiredoxin (2-CysPrx) in the raphidophyte Chattonella marina var. antiqua, a species that induces mass mortality of aquacultured fish. The abundance of the C. marina 2-CysPrx enzyme was highest in the exponential growth phase, during which photosynthetic activity was high, and it then decreased by about a factor of two during the late stationary growth phase. This pattern suggested that 2-CysPrx is a key enzyme involved in the maintenance of high photosynthesis activity. In addition, the fact that the depression of photosynthesis by excessively high irradiance was more severe in the 2-CysPrx low-expression strain (wild type) than in the normal-expression strain (wild type) of C. marina suggested that 2-CysPrx played a critical role in protecting the cell from oxidative stress caused by exposure to excessively high irradiance. In the field of HAB research, estimates of growth potential have been desired to predict the population dynamics of HABs for mitigating damage to fisheries. Therefore, omics approaches have recently begun to be applied to elucidate the physiology of the growth of HAB species. In this review, we describe the progress we have made using a molecular physiological approach to identify the roles of 2-CysPrx and other antioxidant enzymes in mitigating environmental stress associated with strong light and high temperatures and resultant oxidative stress. We also describe results of a survey of expressed Prx genes and their growth-phase-dependent behavior in C. marina using RNA-seq analysis. Finally, we speculate about the function of these genes and the ecological significance of 2-CysPrx, such as its involvement in circadian rhythms and the toxicity of C. marina to fish.

scholarly journals Effects of Saline, an Ambient Acidic Environment, and Sodium Salicylate on OXA-Mediated Carbapenem Resistance in Acinetobacter baumannii: TABLE 1

2016 ◽  
Vol 60 (6) ◽  
pp. 3415-3418 ◽  
Author(s):  
Esther Zander ◽  
Harald Seifert ◽  
Paul G. Higgins
Keyword(s):  
Gene Expression ◽  
Acinetobacter Baumannii ◽  
Sodium Salicylate ◽  
Carbapenem Resistance ◽  
Low Ph ◽  
Wild Type ◽  
Experimental Conditions ◽  
Content Type ◽  
Reference Strains

Different physiological conditions, such as NaCl, low pH, and sodium salicylate, have been shown to affect antibiotic resistance determinants inAcinetobacter baumanniiisolates. Therefore, the aim of this study was to investigate the effects of NaCl, sodium salicylate, and low pH on the susceptibility ofA. baumanniito carbapenem. We cloned genes encoding oxacillinases (OXA) of different subclasses, with their associated promoters, from carbapenem-resistantA. baumanniiisolates into the same vector and transferred them to theA. baumanniireference strains ATCC 19606 and ATCC 17978. Carbapenem MICs were determined at least in triplicate by agar dilution under standard conditions, as well as in the presence of 200 mM NaCl or 16 mM sodium salicylate, or at pH 5.8. OXA-58-like gene expression was determined by reverse transcription-quantitative PCR (qRT-PCR). Under some experimental conditions, significant MIC reductions were shown for some transformants but not for others. Only in one instance were all transformants harboring the same OXA affected by the same condition: at pH 5.8, the imipenem and meropenem MICs for strains expressing OXA-58-like enzymes decreased from a resistant level (32 to 64 mg/liter) to an intermediate-susceptible level (8 mg/liter). However,blaOXA-58-likegene expression remained the same. MICs for both wild-type reference strains were not affected by the conditions tested. Our results indicate that the effects of the experimental conditions tested on OXAin vivoare mostly strain dependent. MICs were not reduced to wild-type levels, suggesting that the conditions tested do not lead to complete OXA inhibition in the bacterial cell.

scholarly journals Synthetic gene regulatory networks in the opportunistic human pathogen Streptococcus pneumoniae

2019 ◽  
Author(s):  
Robin A. Sorg ◽  
Clement Gallay ◽  
Jan-Willem Veening
Keyword(s):  
Gene Expression ◽  
Streptococcus Pneumoniae ◽  
Regulatory Networks ◽  
Expression Patterns ◽  
Combinatorial Libraries ◽  
Regulatory Elements ◽  
Expression Control ◽  
Gene Expression Control

AbstractStreptococcus pneumoniae can cause disease in various human tissues and organs, including the ear, the brain, the blood and the lung, and thus in highly diverse and dynamic environments. It is challenging to study how pneumococci control virulence factor expression, because cues of natural environments and the presence of an immune system are difficult to simulate in vitro. Here, we apply synthetic biology methods to reverse-engineer gene expression control in S. pneumoniae. A selection platform is described that allows for straightforward identification of transcriptional regulatory elements out of combinatorial libraries. We present TetR- and LacI-regulated promoters that show expression ranges of four orders of magnitude. Based on these promoters, regulatory networks of higher complexity are assembled, such as logic AND and IMPLY gates. Finally, we demonstrate single-copy genome-integrated toggle switches that give rise to bimodal population distributions. The tools described here can be used to mimic complex expression patterns, such as the ones found for pneumococcal virulence factors, paving the way for in vivo investigations of the importance of gene expression control on the pathogenicity of S. pneumoniae.

scholarly journals Directed Evolution of an Influenza Reporter Virus To Restore Replication and Virulence and Enhance Noninvasive Bioluminescence Imaging in Mice

2018 ◽  
Vol 92 (16) ◽  
Author(s):  
Hui Cai ◽  
Meisui Liu ◽  
Charles J. Russell
Keyword(s):  
Gene Expression ◽  
Influenza Virus ◽  
Virus Infection ◽  
Directed Evolution ◽  
Influenza Virus Infection ◽  
Foreign Gene ◽  
Reporter Virus ◽  
Wild Type ◽  
Bioluminescence Signal

ABSTRACTReporter viruses provide a powerful tool to study infection, yet incorporating a nonessential gene often results in virus attenuation and genetic instability. Here, we used directed evolution of a luciferase-expressing pandemic H1N1 (pH1N1) 2009 influenza A virus in mice to restore replication kinetics and virulence, increase the bioluminescence signal, and maintain reporter gene expression. An unadapted pH1N1 virus withNanoLuc luciferaseinserted into the 5′ end of the PA gene segment grew to titers 10-fold less than those of the wild type in MDCK cells and in DBA/2 mice and was less virulent. For 12 rounds, we propagated DBA/2 lung samples with the highest bioluminescence-to-titer ratios. Every three rounds, we comparedin vivoreplication, weight loss, mortality, and bioluminescence. Mouse-adapted virus after 9 rounds (MA-9) had the highest relative bioluminescence signal and had wild-type-like fitness and virulence in DBA/2 mice. Using reverse genetics, we discovered fitness was restored in virus rPB2-MA9/PA-D479N by a combination of PA-D479N and PB2-E158G amino acid mutations andPB2noncoding mutations C1161T and C1977T. rPB2-MA9/PA-D479N has increased mRNA transcription, which helps restore wild-type-like phenotypes in DBA/2 and BALB/c mice. Overall, the results demonstrate that directed evolution that maximizes foreign-gene expression while maintaining genetic stability is an effective method to restore wild-type-likein vivofitness of a reporter virus. Virus rPB2-MA9/PA-D479N is expected to be a useful tool for noninvasive imaging of pH1N1 influenza virus infection and clearance while analyzing virus-host interactions and developing new therapeutics and vaccines.IMPORTANCEInfluenza viruses contribute to 290,000 to 650,000 deaths globally each year. Infection is studied in mice to learn how the virus causes sickness and to develop new drugs and vaccines. During experiments, scientists have needed to euthanize groups of mice at different times to measure the amount of infectious virus in mouse tissues. By inserting a foreign gene that causes infected cells to light up, scientists could see infection spread in living mice. Unfortunately, adding an extra gene not needed by the virus slowed it down and made it weaker. Here, we used a new strategy to restore the fitness and lethality of an influenza reporter virus; we adapted it to mouse lungs and selected for variants that had the greatest light signal. The adapted virus can be used to study influenza virus infection, immunology, and disease in living mice. The strategy can also be used to adapt other viruses.

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