Alteration of Salmonella enterica Virulence and Host Pathogenesis through Targeting sdiA by Using the CRISPR-Cas9 System

Salmonella enterica is a common cause of many enteric infections worldwide and is successfully engineered to deliver heterologous antigens to be used as vaccines. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) RNA-guided Cas9 endonuclease is a promising genome editing tool. In the current study, a CRISPR-Cas9 system was used to target S.enterica sdiA that encodes signal molecule receptor SdiA and responds to the quorum sensing (QS) signaling compounds N-acylhomoserine lactones (AHLs). For this purpose, sdiA was targeted in both S.enterica wild type (WT) and the ΔssaV mutant strain, where SsaV has been reported to be an essential component of SPI2-T3SS. The impact of sdiA mutation on S. enterica virulence was evaluated at both early invasion and later intracellular replication in both the presence and absence of AHL. Additionally, the influence of sdiA mutation on the pathogenesis S. enterica WT and mutants was investigated in vivo, using mice infection model. Finally, the minimum inhibitory concentrations (MICs) of various antibiotics against S. enterica strains were determined. Present findings show that mutation in sdiA significantly affects S.enterica biofilm formation, cell adhesion and invasion. However, sdiA mutation did not affect bacterial intracellular survival. Moreover, in vivo bacterial pathogenesis was markedly lowered in S.enterica ΔsdiA in comparison with the wild-type strain. Significantly, double-mutant sdiA and ssaV attenuated the S. enterica virulence and in vivo pathogenesis. Moreover, mutations in selected genes increased Salmonella susceptibility to tested antibiotics, as revealed by determining the MICs and MBICs of these antibiotics. Altogether, current results clearly highlight the importance of the CRISPR-Cas9 system as a bacterial genome editing tool and the valuable role of SdiA in S.enterica virulence. The present findings extend the understanding of virulence regulation and host pathogenesis of Salmonellaenterica.

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Aggregation via the Red, Dry, and Rough Morphotype Is Not a Virulence Adaptation in Salmonella enterica Serovar Typhimurium

Infection and Immunity ◽

10.1128/iai.01383-07 ◽

2008 ◽

Vol 76 (3) ◽

pp. 1048-1058 ◽

Cited By ~ 44

Author(s):

A. P. White ◽

D. L. Gibson ◽

G. A. Grassl ◽

W. W. Kay ◽

B. B. Finlay ◽

...

Keyword(s):

In Vivo Imaging ◽

Salmonella Enterica ◽

Salmonella Enterica Serovar Typhimurium ◽

Primary Role ◽

Wild Type ◽

Serovar Typhimurium ◽

Cell Densities ◽

Two Populations

ABSTRACT The Salmonella rdar (red, dry, and rough) morphotype is an aggregative and resistant physiology that has been linked to survival in nutrient-limited environments. Growth of Salmonella enterica serovar Typhimurium was analyzed in a variety of nutrient-limiting conditions to determine whether aggregation would occur at low cell densities and whether the rdar morphotype was involved in this process. The resulting cultures consisted of two populations of cells, aggregated and nonaggregated, with the aggregated cells preferentially displaying rdar morphotype gene expression. The two groups of cells could be separated based on the principle that aggregated cells were producing greater amounts of thin aggregative fimbriae (Tafi or curli). In addition, the aggregated cells retained some physiological characteristics of the rdar morphotype, such as increased resistance to sodium hypochlorite. Competitive infection experiments in mice showed that nonaggregative ΔagfA cells outcompeted rdar-positive wild-type cells in all tissues analyzed, indicating that aggregation via the rdar morphotype was not a virulence adaptation in Salmonella enterica serovar Typhimurium. Furthermore, in vivo imaging experiments showed that Tafi genes were not expressed during infection but were expressed once Salmonella was passed out of the mice into the feces. We hypothesize that the primary role of the rdar morphotype is to enhance Salmonella survival outside the host, thereby aiding in transmission.

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The Role of graRS in Regulating Virulence and Antimicrobial Resistance in Methicillin-Resistant Staphylococcus aureus

Frontiers in Microbiology ◽

10.3389/fmicb.2021.727104 ◽

2021 ◽

Vol 12 ◽

Author(s):

Le Chen ◽

Zihui Wang ◽

Tao Xu ◽

Hongfei Ge ◽

Fangyue Zhou ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Type Strain ◽

Bacterial Resistance ◽

Wild Type Strain ◽

Lysine Biosynthesis ◽

Infection Model ◽

Wild Type ◽

Methicillin Resistant

Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of both community- and hospital-associated infections. The antibiotic resistance and virulence characteristics of MRSA are largely regulated by two-component signal transduction systems (TCS) including the graRS TCS. To make a relatively comprehensive insight into graRS TCS in MRSA, the bioinformatics analysis of dataset GSE26016 (a S. aureus HG001 WT strain vs. the ΔgraRS mutant) from Gene Expression Omnibus (GEO) database was performed, and a total of 563 differentially expressed genes (DEGs) were identified. GO analysis revealed that the DEGs were mainly enriched in the “de novo” IMP biosynthetic process, lysine biosynthetic process via diaminopimelate, and pathogenesis; and they were mainly enriched in purine metabolism, lysine biosynthesis, and monobactam biosynthesis in KEGG analysis. WGCNA suggested that the turquoise module was related to the blue module, and the genes in these two modules were associated with S. aureus virulence and infection. To investigate the role of graRS in bacterial virulence, a graRS knockout mutant (ΔgraRS) was constructed using MRSA USA500 2,395 strain as a parent strain. Compared to the wild-type strain, the USA500ΔgraRS showed reduced staphyloxanthin production, retarded coagulation, weaker hemolysis on blood agar plates, and a decreased biofilm formation. These altered phenotypes were restored by the complementation of a plasmid-expressed graRS. Meanwhile, an expression of the virulence-associated genes (coa, hla, hlb, agrA, and mgrA) was downregulated in the ΔgraRS mutant. Consistently, the A549 epithelial cells invasion of the ΔgraRS mutant was 4-fold lower than that of the USA500 wild-type strain. Moreover, on the Galleria mellonella infection model, the survival rate at day 5 post infection in the USA500ΔgraRS group (55%) was obviously higher than that in the USA500 group (20%), indicating graRS knockout leads to a decreased virulence in vivo. In addition, the deletion of the graRS in the MRSA USA500 strain resulted in its increased susceptibilities to ampicillin, oxacillin, vancomycin, and gentamicin. Our work suggests that the graRS TCS plays an important role in regulating S. aureus virulence in vitro and in vivo and modulate bacterial resistance to various antibiotics.

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In VivoActivities of Ceftolozane, a New Cephalosporin, with and without Tazobactam against Pseudomonas aeruginosa and Enterobacteriaceae, Including Strains with Extended-Spectrum β-Lactamases, in the Thighs of Neutropenic Mice

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01590-12 ◽

2012 ◽

Vol 57 (4) ◽

pp. 1577-1582 ◽

Cited By ~ 88

Author(s):

W. A. Craig ◽

D. R. Andes

Keyword(s):

Pseudomonas Aeruginosa ◽

Infection Model ◽

Wild Type ◽

Gram Negative ◽

Content Type ◽

Peak Dose ◽

Extended Spectrum ◽

The Impact ◽

Primary Index

ABSTRACTCeftolozane is a new cephalosporin with potent activity againstPseudomonas aeruginosaandEnterobacteriaceae. A neutropenic murine thigh infection model was used to determine which pharmacokinetic/pharmacodynamic index and magnitude drives the efficacy of ceftolozane with Gram-negative bacilli, to compare the rates ofin vivokilling ofP. aeruginosaby ceftolozane and ceftazidime, and to determine the impact of different ratios of ceftolozane plus tazobactam onEnterobacteriaceaecontaining extended-spectrum β-lactamases (ESBLs). Neutropenic mice had 106.2-7.1CFU/thigh when treated with ceftolozane for 24 h with (i) various doses (3.12 to 1,600 mg/kg) and dosage intervals (3, 6, 12, and 24 h) against twoEnterobacteriaceaestrains, (ii) 0.39 to 800 mg/kg every 6 h for fourEnterobacteriaceaeand fourP. aeruginosastrains, and (iii) 400 or 800 mg/kg with 2:1. 4:1, and 8:1 ratios of tazobactam against fiveEnterobacteriaceaestrains with ESBLs. The pharmacokinetics of ceftolozane at 25, 100, and 400 mg/kg were linear with peak/dose values of 1.0 to 1.4 and half-lives of 12 to 14 min. T>MIC was the primary index driving efficacy. For stasis (1 log kill), T>MIC was 26.3% ± 2.1% (31.6% ± 1.6%) for wild-typeEnterobacteriaceae, 31.1% ± 4.9% (34.8% ± 4.4%) forEnterobacteriaceaewith ESBLs, and 24.0% ± 3.3% (31.5% ± 3.9%) forP. aeruginosa. At 200 mg/kg every 3 h, the rate ofin vivokilling ofP. aeruginosawas faster with ceftolozane than with ceftazidime (−0.34 to −0.41 log10CFU/thigh/h versus −0.21 to −0.24 log10CFU/thigh/h). The 2:1 ratio of ceftolozane with tazobactam was the most potent combination studied. The T>MIC required for ceftolozane is less than with other cephalosporins and may be due to more rapid killing.

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Analysis of Spleen-Induced Fimbria Production in Recombinant AttenuatedSalmonella entericaSerovar Typhimurium Vaccine Strains

mBio ◽

10.1128/mbio.01189-17 ◽

2017 ◽

Vol 8 (4) ◽

Cited By ~ 10

Author(s):

Paweł Łaniewski ◽

Chang-Ho Baek ◽

Kenneth L. Roland ◽

Roy Curtiss

Keyword(s):

Salmonella Enterica ◽

Protective Efficacy ◽

The United States ◽

Wild Type ◽

Strong Immune Response ◽

Food Borne ◽

Serovar Typhimurium ◽

Oral Doses

ABSTRACTSalmonella entericaserovar Typhimurium genome encodes 13 fimbrial operons. Most of the fimbriae encoded by these operons are not produced under laboratory conditions but are likely to be synthesizedin vivo. We used anin vivoexpression technology (IVET) strategy to identify four fimbrial operons,agf,saf,sti, andstcthat are expressed in the spleen. When any three of these operons were deleted, the strain retained wild-type virulence. However, when all four operons were deleted, the resulting strain was completely attenuated, indicating that these four fimbriae play functionally redundant roles critical for virulence. In mice, oral doses of as low as 1 × 105 CFU of the strain with four fimbrial operons deleted provided 100% protection against challenge with 1 × 109 CFU of wild-typeS. Typhimurium. We also examined the possible effect of these fimbriae on the ability of aSalmonellavaccine strain to deliver a guest antigen. We modified one of our established attenuated vaccine strains, χ9088, to delete three fimbrial operons while the fourth operon was constitutively expressed. Each derivative was modified to express theStreptococcus pneumoniaeantigen PspA. Strains that constitutively expressedsaforstcelicited a strong Th1 response with significantly greater levels of anti-PspA serum IgG and greater protective efficacy than strains carryingsaforstcdeletions. The isogenic strain in which all four operons were deleted generated the lowest anti-PspA levels and did not protect against challenge with virulentS. pneumoniae. Our results indicate that these fimbriae play important roles, as yet not understood, inSalmonellavirulence and immunogenicity.IMPORTANCESalmonella entericais the leading cause of bacterial food-borne infection in the United States. S. Typhimurium is capable of producing up to 13 distinct surface structures called fimbriae that presumably mediate its adherence to surfaces. The roles of most of these fimbriae in disease are unknown. Identifying fimbriae produced during infection will provide important insights into how these bacterial structures contribute to disease and potentially induce protective immunity toSalmonellainfection. We identified four fimbriae that are produced during infection. Deletion of all four of these fimbriae results in a significant reduction in virulence. We explored ways in which the expression of these fimbriae may be exploited for use in recombinantSalmonellavaccine strains and found that production of Saf and Stc fimbriae are important for generating a strong immune response against a vectored antigen. This work provides new insight into the role of fimbriae in disease and their potential for improving the efficacy ofSalmonella-based vaccines.

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Salmonella enterica Serovar Typhimurium HtrA: regulation of expression and role of the chaperone and protease activities during infection

Microbiology ◽

10.1099/mic.0.023754-0 ◽

2009 ◽

Vol 155 (3) ◽

pp. 873-881 ◽

Cited By ~ 42

Author(s):

Claire Lewis ◽

Henrieta Skovierova ◽

Gary Rowley ◽

Bronislava Rezuchova ◽

Dagmar Homerova ◽

...

Keyword(s):

Salmonella Enterica ◽

Protease Activity ◽

Pdz Domain ◽

Proximal Promoter ◽

Wild Type ◽

Regulation Of Expression ◽

Serovar Typhimurium

HtrA is a bifunctional stress protein required by many bacterial pathogens to successfully cause infection. Salmonella enterica serovar Typhimurium (S. Typhimurium) htrA mutants are defective in intramacrophage survival and are highly attenuated in mice. Transcription of htrA in Escherichia coli is governed by a single promoter that is dependent on σ E (RpoE). S. Typhimurium htrA also possesses a σ E-dependent promoter; however, we found that the absence of σ E had little effect on production of HtrA by S. Typhimurium. This suggests that additional promoters control expression of htrA in S. Typhimurium. We identified three S. Typhimurium htrA promoters. Only the most proximal promoter, htrAp3, was σ E dependent. The other promoters, htrAp1 and htrAp2, are probably recognized by the principal sigma factor σ 70. These two promoters were constitutively expressed but were also slightly induced by heat shock. Thus expression of htrA is different in S. Typhimurium and E. coli. The role of HtrA is to deal with misfolded/damaged proteins in the periplasm. It can do this either by degrading (protease activity) or folding/capturing (chaperone/sequestering, C/S, activity) the aberrant protein. We investigated which of these functions are important to S. Typhimurium in vitro and in vivo. Point or deletion mutants of htrA that encode variant HtrA molecules have been used in previous studies to investigate the role of different regions of HtrA in C/S and protease activity. These htrA variants were placed under the control of the S. Typhimurium htrAP123 promoters and expressed in a S. Typhimurium htrA mutant, GVB1343. Both wild-type HtrA and HtrA (HtrA S210A) lacking protease activity enabled GVB1343 to grow at high temperature (46 °C). Both molecules also significantly enhanced the growth/survival of GVB1343 in the liver and spleen of mice during infection. However, expression of wild-type HtrA enabled GVB1343 to grow to much higher levels than expression of HtrA S210A. Thus both the protease and C/S functions of HtrA operate in vivo during infection but the protease function is probably more important. Absence of either PDZ domain completely abolished the ability of HtrA to complement the growth defects of GVB1343 in vitro or in vivo.

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Evaluation of Regulated Delayed Attenuation Strategies for Salmonella enterica Serovar Typhi Vaccine Vectors in Neonatal and Infant Mice

Clinical and Vaccine Immunology ◽

10.1128/cvi.00003-13 ◽

2013 ◽

Vol 20 (6) ◽

pp. 931-944 ◽

Cited By ~ 9

Author(s):

Huoying Shi ◽

Shifeng Wang ◽

Roy Curtiss

Keyword(s):

Salmonella Enterica ◽

Peyer's Patches ◽

Interleukin 4 ◽

Peyer’S Patches ◽

Wild Type ◽

Vaccine Vectors ◽

Content Type ◽

The Impact

ABSTRACTWe developed regulated delayed attenuation strategies forSalmonellavaccine vectors. In this study, we evaluated the combination of these strategies in recombinant attenuatedSalmonella entericaserovar Typhi andSalmonella entericaserovar Typhimurium vaccine vectors with similar genetic backgroundsin vitroandin vivo. Our goal is to develop a vaccine to preventStreptococcus pneumoniaeinfection in newborns; thus, all strains delivered a pneumococcal antigen PspA and the impact of maternal antibodies was evaluated. The results showed that all strains with the regulated delayed attenuated phenotype (RDAP) displayed an invasive ability stronger than that of theS.Typhi vaccine strain, Ty21a, but weaker than that of their corresponding wild-type parental strains. The survival curves of different RDAP vaccine vectorsin vitroandin vivoexhibited diverse regulated delayed attenuation kinetics, which was different fromS.Typhi Ty21a and the wild-type parental strains. Under the influence of maternal antibody, the persistence of theS.Typhimurium RDAP strain displayed a regulated delayed attenuation trend in nasal lymphoid tissue (NALT), lung, and Peyer's patches, while the persistence ofS.Typhi RDAP strains followed the curve only in NALT. The bacterial loads ofS.Typhi RDAP strains were lower in NALT, lung, and Peyer's patches in mice born to immune mothers than in those born to naive mothers. In accordance with these results, RDAP vaccine strains induced high titers of IgG antibodies against PspA and againstSalmonellalipopolysaccharides. Immunization of mothers withS.Typhi RDAP strains enhanced the level of vaginal mucosal IgA, gamma interferon (IFN-γ), and interleukin 4 (IL-4) and resulted in a higher level of protection againstS. pneumoniaechallenge.

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Induction of Cyclooxygenase 2 by Streptococcus pyogenes Is Mediated by Cytolysins

Journal of Innate Immunity ◽

10.1159/000479153 ◽

2017 ◽

Vol 9 (6) ◽

pp. 587-597 ◽

Cited By ~ 4

Author(s):

Ulrike Blaschke ◽

Andreas Beineke ◽

Johanna Klemens ◽

Eva Medina ◽

Oliver Goldmann

Keyword(s):

Streptococcus Pyogenes ◽

Cyclooxygenase 2 ◽

Arachidonic Acid Metabolite ◽

Infection Model ◽

Synergistic Activity ◽

Wild Type ◽

Cox 2

Prostaglandin E2 (PGE2), an arachidonic acid metabolite regulating a broad range of physiological activities, is an important modulator of the severity of infection caused by Streptococcus pyogenes. Here, we investigated the role of streptococcal cytolysin S (SLS) and streptococcal cytolysin O (SLO) in the induction of cyclooxygenase-2 (COX-2), the rate-limiting enzyme in the synthesis of prostaglandins, in in vitro cultured macrophages and during in vivo infection. Macrophages were infected with S. pyogenes wild type or with the isogenic mutant strains deficient in SLS (ΔSLS), SLO (ΔSLO), or both (ΔSLS/ΔSLO), and the expression of COX-2 was determined at the transcriptional and the protein level. The results indicated that S. pyogenes induced expression of COX-2 and concomitant synthesis of PGE2 in macrophages mediated by the synergistic activity of both SLS and SLO, and involved calcium and the PKC/JNK signaling pathway. These results were validated using recombinant cytolysins. In a murine skin infection model, COX-2-positive cells were found more abundant at the site of S. pyogenes wild-type infection than at the site of infection with ΔSLS/ΔSLO mutant strain. These findings suggest that inhibitory targeting of SLS and SLO could ameliorate the adverse effects of high levels of prostaglandins during S. pyogenes infection.

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Role of Energy Sensor TlpD of Helicobacter pylori in Gerbil Colonization and Genome Analyses after Adaptation in the Gerbil

Infection and Immunity ◽

10.1128/iai.00750-13 ◽

2013 ◽

Vol 81 (10) ◽

pp. 3534-3551 ◽

Cited By ~ 25

Author(s):

Wiebke Behrens ◽

Tobias Schweinitzer ◽

Joena Bal ◽

Martina Dorsch ◽

André Bleich ◽

...

Keyword(s):

Helicobacter Pylori ◽

Human Isolate ◽

Infection Model ◽

Energy Yield ◽

Content Type ◽

Full Characterization ◽

H Pylori ◽

The Impact

ABSTRACTHelicobacter pylorimaintains colonization in its human host using a limited set of taxis sensors. TlpD is a proposed energy taxis sensor ofH. pyloriand dominant under environmental conditions of low bacterial energy yield. We studied the impact ofH. pyloriTlpD on colonizationin vivousing a gerbil infection model which closely mimics the gastric physiology of humans. A gerbil-adaptedH. pyloristrain, HP87 P7, showed energy-dependent behavior, while its isogenictlpDmutant lost it. A TlpD-complemented strain regained the wild-type phenotype. Infection of gerbils with the complemented strain demonstrated that TlpD is important for persistent infection in the antrum and corpus and suggested a role of TlpD in horizontal navigation and persistent corpus colonization. As a part of the full characterization of the model and to gain insight into the genetic basis ofH. pyloriadaptation to the gerbil, we determined the complete genome sequences of the gerbil-adapted strain HP87 P7, two HP87 P7tlpDmutants before and after gerbil passage, and the original human isolate, HP87. The integrity of the genome, including that of a functionalcagpathogenicity island, was maintained after gerbil adaptation. Genetic and phenotypic differences between the strains were observed. Major differences between the gerbil-adapted strain and the human isolate emerged, including evidence of recent recombination. Passage of thetlpDmutant through the gerbil selected for gain-of-function variation in a fucosyltransferase gene,futC(HP0093). In conclusion, a gerbil-adaptedH. pyloristrain with a stable genome has helped to establish that TlpD has important functions for persistent colonization in the stomach.

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Role of GIRK channels on the noradrenergic transmission in vivo: an electrophysiological and neurochemical study on GIRK2 mutant mice

The International Journal of Neuropsychopharmacology ◽

10.1017/s1461145712000971 ◽

2013 ◽

Vol 16 (5) ◽

pp. 1093-1104 ◽

Cited By ~ 8

Author(s):

María Torrecilla ◽

Irrintzi Fernández-Aedo ◽

Aurora Arrue ◽

Mercedes Zumarraga ◽

Luisa Ugedo

Keyword(s):

Mutant Mice ◽

Wild Type ◽

Girk Channels ◽

Noradrenergic Transmission ◽

Homozygous Genotype ◽

The Impact ◽

G Protein Coupled

Abstract Dysfunctional noradrenergic transmission is related to several neuropsychiatric conditions, such as depression. Nowadays, the role of G protein-coupled inwardly rectifying potassium (GIRK)2 subunit containing GIRK channels controlling neuronal intrinsic excitability in vitro is well known. The aim of this study was to investigate the impact of GIRK2 subunit mutation on the central noradrenergic transmission in vivo. For that purpose, single-unit extracellular activity of locus coeruleus (LC) noradrenergic neurons and brain monoamine levels using the HPLC technique were measured in wild-type and GIRK2 mutant mice. Girk2 gene mutation induced significant differences among genotypes regarding burst activity of LC neurons. In fact, the proportion of neurons displaying burst firing was increased in GIRK2 heterozygous mice as compared to that recorded from wild-type mice. Furthermore, this augmentation was even greater in the homozygous genotype. However, neither the basal firing rate nor the coefficient of variation of LC neurons was different among genotypes. Noradrenaline and serotonin basal levels were altered in the dorsal raphe nucleus from GIRK2 heterozygous and homozygous mice, respectively. Furthermore, noradrenaline levels were increased in LC projecting areas such as the hippocampus and amygdale from homozygous mice, although not in the prefrontal cortex. Finally, potency of clonidine and morphine inhibiting LC activity was reduced in GIRK2 mutant mice, although the efficacy remained unchanged. Altogether, the present study supports the role of GIRK2 subunit-containing GIRK channels on the maintenance of tonic noradrenergic activity in vivo. Electric and neurochemical consequences derived from an altered GIRK2-dependent signalling could facilitate the understanding of the neurobiological basis of pathologies related to a dysfunctional monoaminergic transmission.

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Deciphering the role of ttrA and pduA genes for Salmonella enterica serovars in a chicken infection model

Avian Pathology ◽

10.1080/03079457.2021.1909703 ◽

2021 ◽

pp. 1-41

Author(s):

MMS Saraiva ◽

LB Rodrigues Alves ◽

DFM Monte ◽

TS Ferreira ◽

VP Benevides ◽

...

Keyword(s):

Salmonella Enterica ◽

Infection Model

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