Protective Effect of Nasal Colonisation with ∆cps/piaA and ∆cps/proABCStreptococcus pneumoniae Strains against Recolonisation and Invasive Infection

Rationale: Nasopharyngeal administration of live virulence-attenuated Streptococcus pneumoniae strains is a potential novel preventative strategy. One target for creating reduced virulence S. pneumoniae strains is the capsule, but loss of the capsule reduces the duration of S. pneumoniae colonisation in mice which could impair protective efficacy against subsequent infection. Objectives: To assess protective efficacy of nasopharyngeal administration of unencapsulated S. pneumoniae strains in murine infection models. Methods: Strains containing cps locus deletions combined with the S. pneumoniae virulence factors psaA (reduces colonisation) or proABC (no effect on colonisation) were constructed and their virulence phenotypes and ability to prevent recolonisation or invasive infection assessed using mouse infection models. Serological responses to colonisation were compared between strains using ELISAs, immunoblots and 254 S. pneumoniae protein antigen array. Measurements and Main Results: The ∆cps/piaA and ∆cps/proABC strains were strongly attenuated in virulence in both invasive infection models and had a reduced ability to colonise the nasopharynx. ELISAs, immunoblots and protein arrays showed colonisation with either strain stimulated weaker serological responses than the wild type strain. Mice previously colonised with these strains were protected against septicaemic pneumonia but, unlike mice colonised with the wild type strain, not against S. pneumoniae recolonisation. Conclusions: Colonisation with the ∆cps/piaA and ∆cps/proABC strains prevented subsequent septicaemia, but in contrast, to published data for encapsulated double mutant strains they did not prevent recolonisation with S. pneumoniae. These data suggest targeting the cps locus is a less effective option for creating live attenuated strains that prevent S. pneumoniae infections.

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Characterization and Evaluation of a Salmonella enterica Serotype Senftenberg Mutant Created by Deletion of Virulence-Related Genes for Use as a Live Attenuated Vaccine

Clinical and Vaccine Immunology ◽

10.1128/cvi.00233-16 ◽

2016 ◽

Vol 23 (10) ◽

pp. 802-812 ◽

Cited By ~ 5

Author(s):

Nitin M. Kamble ◽

John Hwa Lee

Keyword(s):

Salmonella Enterica ◽

Type Strain ◽

Wild Type Strain ◽

Protective Efficacy ◽

Control Group ◽

Intestinal Loop ◽

Wild Type ◽

Exchange Method ◽

Content Type

ABSTRACTNatural infections of chickens withSalmonella entericasubsp.entericaserovar Senftenberg (S.Senftenberg) are characterized by low-level intestinal invasiveness and insignificant production of antibodies. In this study, we investigated the potential effects oflonandcpxRgene deletions on the invasiveness ofS. Senftenberg into the intestinal epithelium of chickens and its ability to induce an immune response, conferring protection againstS. Senftenberg infection. With the allelic exchange method, we developed JOL1596 (Δlon), JOL1571 (ΔcpxR), and JOL1587 (ΔlonΔcpxR) deletion mutants from wild-typeS. Senftenberg. Deletion of thelongene fromS. Senftenberg produced increased frequency of elongated cells, with significantly greater amounts of exopolysaccharide (EPS) than in thecpxR-deleted strain and the wild-type strain. Thein vivointestinal loop invasion assay showed a significant increase in epithelial invasiveness for JOL1596 (Δlon) and JOL1587 (ΔlonΔcpxR), compared to JOL1571 (ΔcpxR) and the wild-type strain. Furthermore, theS. Senftenberg wild-type and mutant strains were internalized at high levels inside activated abdominal macrophages from chicken. Thein vivoinoculation of JOL1587 (ΔlonΔcpxR) into chickens led to colonization of the liver, spleen, and cecum for a short time. Chickens inoculated with JOL1587 (ΔlonΔcpxR) showed significant increases in humoral, mucosal, and cellular immune responses specific toS. Senftenberg antigens. Postchallenge, compared to the control group, the JOL1587 (ΔlonΔcpxR)-inoculated chickens showed not only lower persistence but also faster clearance of wild-typeS. Senftenberg from the cecum. We conclude that the increased intestinal invasiveness and colonization of internal organs exhibited by JOL1587 (ΔlonΔcpxR) led to the establishment of immunogenicity and conferred protective efficacy againstS. Senftenberg infections in chickens.

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Absence of a Cysteine Protease Effect on Bacterial Virulence in Two Murine Models of Human Invasive Group A Streptococcal Infection

Infection and Immunity ◽

10.1128/iai.69.11.6683-6686.2001 ◽

2001 ◽

Vol 69 (11) ◽

pp. 6683-6688 ◽

Cited By ~ 33

Author(s):

Cameron D. Ashbaugh ◽

Michael R. Wessels

Keyword(s):

Cysteine Protease ◽

Type Strain ◽

Wild Type Strain ◽

Streptococcal Infection ◽

Invasive Infection ◽

Protease Gene ◽

Wild Type ◽

Phenotypic Properties ◽

Mutant Strains ◽

Group A

ABSTRACT The cysteine protease of group A streptococci has been suggested to contribute to the pathogenesis of invasive infection through degradation of host tissue, activation of the host inflammatory response, release of protective molecules from the bacterial cell surface, or other mechanisms. However, studies of the effects on virulence of inactivating the cysteine protease genespeB have yielded conflicting results. In some reports, a speB mutant was relatively avirulent in mouse models of invasive infection whereas little or no attenuation of virulence was observed in other studies of similar mutant strains. Possible reasons for these discordant results include differences in the streptococcal strains from which the speB mutants were derived, differences in the infection models employed, or unintended effects on another virulence determinant(s) that arose during the derivation of aspeB mutant. We attempted to clarify these issues by characterizing the phenotypic properties and relative virulence in mice of two speB mutant strains, both derived from wild-type strain AM3: speB mutant AM3speB, which has been shown to be markedly attenuated in virulence in mice after intraperitoneal or subcutaneous challenge, and AM3speBΩ, a new mutant strain derived for this investigation. Both mutant strains were negative for protease activity, as expected, and both produced wild-type amounts of type 3 M protein and streptolysin O. However, AM3speB produced significantly less cell-associated hyaluronic acid capsule than did parent strain AM3 or strain AM3speBΩ. Compared to wild-type strain AM3, AM3speB was more sensitive to opsonophagocytic killing in vitro and was significantly less virulent in mice after intraperitoneal challenge. By contrast, AM3speBΩ was fully resistant to phagocytosis and did not differ significantly from the wild-type strain in mouse virulence after an intraperitoneal or subcutaneous challenge. We concluded that previous reports attributing loss of virulence in strain AM3speB to inactivation of speB are in error. Within the limitations of the models used, we found no effect of cysteine protease on invasive streptococcal infection.

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Effects of Mutations of RAD50, RAD51, RAD52, and Related Genes on Illegitimate Recombination in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/142.2.383 ◽

1996 ◽

Vol 142 (2) ◽

pp. 383-391 ◽

Cited By ~ 2

Author(s):

Yasumasa Tsukamoto ◽

Jun-ichi Kato ◽

Hideo Ikeda

Keyword(s):

Saccharomyces Cerevisiae ◽

Quantitative Analysis ◽

Structural Analysis ◽

Recombination Rate ◽

Type Strain ◽

Negative Selection ◽

Wild Type Strain ◽

Illegitimate Recombination ◽

Wild Type ◽

In The Wild

Abstract To examine the mechanism of illegitimate recombination in Saccharomyces cerevisiae, we have developed a plasmid system for quantitative analysis of deletion formation. A can1 cyh2 cell carrying two negative selection markers, the CAN1 and CYH2 genes, on a YCp plasmid is sensitive to canavanine and cycloheximide, but the cell becomes resistant to both drugs when the plasmid has a deletion over the CAN1 and CYH2 genes. Structural analysis of the recombinant plasmids obtained from the resistant cells showed that the plasmids had deletions at various sites of the CAN1-CYH2 region and there were only short regions of homology (1-5 bp) at the recombination junctions. The results indicated that the deletion detected in this system were formed by illegitimate recombination. Study on the effect of several rad mutations showed that the recombination rate was reduced by 30-, 10-, 10-, and 10-fold in the rad52, rad50, mre11, and xrs2 mutants, respectively, while in the rud51, 54, 55, and 57 mutants, the rate was comparable to that in the wild-type strain. The rad52 mutation did not affect length of homology at junction sites of illegitimate recombination.

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The role of Zur-regulated lipoprotein A in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles in Acinetobacter baumannii

BMC Microbiology ◽

10.1186/s12866-020-02083-0 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Nayeong Kim ◽

Hyo Jeong Kim ◽

Man Hwan Oh ◽

Se Yeon Kim ◽

Mi Hyun Kim ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Outer Membrane ◽

Mutant Strain ◽

Antimicrobial Susceptibility ◽

Type Strain ◽

Wild Type Strain ◽

Membrane Vesicles ◽

Outer Membrane Vesicles ◽

Wild Type ◽

Bacterial Morphology

Abstract Background Zinc uptake-regulator (Zur)-regulated lipoprotein A (ZrlA) plays a role in bacterial fitness and overcoming antimicrobial exposure in Acinetobacter baumannii. This study further characterized the zrlA gene and its encoded protein and investigated the roles of the zrlA gene in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles (OMVs) in A. baumannii ATCC 17978. Results In silico and polymerase chain reaction analyses showed that the zrlA gene was conserved among A. baumannii strains with 97–100% sequence homology. Recombinant ZrlA protein exhibited a specific enzymatic activity of D-alanine-D-alanine carboxypeptidase. Wild-type A. baumannii exhibited more morphological heterogeneity than a ΔzrlA mutant strain during stationary phase. The ΔzrlA mutant strain was more susceptible to gentamicin than the wild-type strain. Sizes and protein profiles of OMVs were similar between the wild-type and ΔzrlA mutant strains, but the ΔzrlA mutant strain produced 9.7 times more OMV particles than the wild-type strain. OMVs from the ΔzrlA mutant were more cytotoxic in cultured epithelial cells than OMVs from the wild-type strain. Conclusions The present study demonstrated that A. baumannii ZrlA contributes to bacterial morphogenesis and antimicrobial resistance, but its deletion increases OMV production and OMV-mediated host cell cytotoxicity.

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FixJ family regulator AcfR of Azorhizobium caulinodans is involved in symbiosis with the host plant

BMC Microbiology ◽

10.1186/s12866-021-02138-w ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Wei Liu ◽

Xue Bai ◽

Yan Li ◽

Haikun Zhang ◽

Xiaoke Hu

Keyword(s):

Signal Transduction ◽

Host Plant ◽

Type Strain ◽

Wild Type Strain ◽

Response Regulator ◽

Azorhizobium Caulinodans ◽

Wild Type ◽

Response Regulators ◽

Free Living ◽

Two Component

Abstract Background A wide variety of bacterial adaptative responses to environmental conditions are mediated by signal transduction pathways. Two-component signal transduction systems are one of the predominant means used by bacteria to sense the signals of the host plant and adjust their interaction behaviour. A total of seven open reading frames have been identified as putative two-component response regulators in the gram-negative nitrogen-fixing bacteria Azorhizobium caulinodans ORS571. However, the biological functions of these response regulators in the symbiotic interactions between A. caulinodans ORS571 and the host plant Sesbania rostrata have not been elucidated to date. Results In this study, we identified and investigated a two-component response regulator, AcfR, with a phosphorylatable N-terminal REC (receiver) domain and a C-terminal HTH (helix-turn-helix) LuxR DNA-binding domain in A. caulinodans ORS571. Phylogenetic analysis showed that AcfR possessed close evolutionary relationships with NarL/FixJ family regulators. In addition, six histidine kinases containing HATPase_c and HisKA domains were predicted to interact with AcfR. Furthermore, the biological function of AcfR in free-living and symbiotic conditions was elucidated by comparing the wild-type strain and the ΔacfR mutant strain. In the free-living state, the cell motility behaviour and exopolysaccharide production of the ΔacfR mutant were significantly reduced compared to those of the wild-type strain. In the symbiotic state, the ΔacfR mutant showed a competitive nodule defect on the stems and roots of the host plant, suggesting that AcfR can provide A. caulinodans with an effective competitive ability for symbiotic nodulation. Conclusions Our results showed that AcfR, as a response regulator, regulates numerous phenotypes of A. caulinodans under the free-living conditions and in symbiosis with the host plant. The results of this study help to elucidate the involvement of a REC + HTH_LuxR two-component response regulator in the Rhizobium-host plant interaction.

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Mab_3083c Is a Homologue of RNase J and Plays a Role in Colony Morphotype, Aggregation, and Sliding Motility of Mycobacterium abscessus

Microorganisms ◽

10.3390/microorganisms9040676 ◽

2021 ◽

Vol 9 (4) ◽

pp. 676

Author(s):

Ting-Yu Liu ◽

Sheng-Hui Tsai ◽

Jenn-Wei Chen ◽

Yu-Ching Wang ◽

Shiau-Ting Hu ◽

...

Keyword(s):

Type Strain ◽

Wild Type Strain ◽

Opportunistic Pathogen ◽

Mycobacterium Abscessus ◽

Colony Morphology ◽

Clinical Strain ◽

Immunocompromised Patients ◽

Clinical Infection ◽

Wild Type ◽

Sliding Motility

Mycobacterium abscessus is an opportunistic pathogen causing human diseases, especially in immunocompromised patients. M. abscessus strains with a rough morphotype are more virulent than those with a smooth morphotype. Morphotype switch may occur during a clinical infection. To investigate the genes involved in colony morphotype switching, we performed transposon mutagenesis in a rough clinical strain of M. abscessus. A morphotype switching mutant (smooth) named mab_3083c::Tn was obtained. This mutant was found to have a lower aggregative ability and a higher sliding motility than the wild type strain. However, its glycopeptidolipid (GPL) content remained the same as those of the wild type. Complementation of the mutant with a functional mab_3083c gene reverted its morphotype back to rough, indicating that mab_3083c is associated with colony morphology of M. abscessus. Bioinformatic analyses showed that mab_3083c has a 75.4% identity in amino acid sequence with the well-characterized ribonuclease J (RNase J) of M. smegmatis (RNase JMsmeg). Complementation of the mutant with the RNase J gene of M. smegmatis also switched its colony morphology from smooth back to rough. These results suggest that Mab_3083c is a homologue of RNase J and involved in regulating M. abscessus colony morphotype switching.

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Mutations of folC cause increased susceptibility to sulfamethoxazole in Mycobacterium tuberculosis

Scientific Reports ◽

10.1038/s41598-020-80213-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ruiqi Wang ◽

Kun Li ◽

Jifang Yu ◽

Jiaoyu Deng ◽

Yaokai Chen

Keyword(s):

Mycobacterium Tuberculosis ◽

Type Strain ◽

Wild Type Strain ◽

Clinical Isolates ◽

Aminobenzoic Acid ◽

Wild Type ◽

Dihydropteroate Synthase ◽

Expression Levels ◽

Encoding Gene ◽

Increased Susceptibility

AbstractPrevious studies showed that mutation of folC caused decreased expression of the dihydropteroate synthase encoding gene folP2 in Mycobacterium tuberculosis (M. tuberculosis). We speculated that mutation of folC in M. tuberculosis might affect the susceptibility to sulfamethoxazole (SMX). To prove this, 53 clinical isolates with folC mutations were selected and two folC mutants (I43A, I43T) were constructed based on M. tuberculosis H37Ra. The results showed that 42 of the 53 clinical isolates (79.2%) and the two lab-constructed folC mutants were more sensitive to SMX. To probe the mechanism by which folC mutations make M. tuberculosis more sensitive to SMX, folP2 was deleted in H37Ra, and expression levels of folP2 were compared between H37Ra and the two folC mutants. Although deletion of folP2 resulted in increased susceptibility to SMX, no difference in folP2 expression was observed. Furthermore, production levels of para-aminobenzoic acid (pABA) were compared between the folC mutants and the wild-type strain, and results showed that folC mutation resulted in decreased production of pABA. Taken together, we show that folC mutation leads to decreased production of pABA in M. tuberculosis and thus affects its susceptibility to SMX, which broadens our understanding of mechanisms of susceptibilities to antifolates in this bacterium.

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Expression and function of the cdgD gene, encoding a CHASE–PAS-DGC-EAL domain protein, in Azospirillum brasilense

Scientific Reports ◽

10.1038/s41598-020-80125-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

José Francisco Cruz-Pérez ◽

Roxana Lara-Oueilhe ◽

Cynthia Marcos-Jiménez ◽

Ricardo Cuatlayotl-Olarte ◽

María Luisa Xiqui-Vázquez ◽

...

Keyword(s):

Azospirillum Brasilense ◽

Type Strain ◽

Wild Type Strain ◽

Soil Conditions ◽

Wild Type ◽

Gene Encoding ◽

Wheat Roots ◽

Genes Encoding ◽

In The Wild ◽

Plant Growth Promoting

AbstractThe plant growth-promoting bacterium Azospirillum brasilense contains several genes encoding proteins involved in the biosynthesis and degradation of the second messenger cyclic-di-GMP, which may control key bacterial functions, such as biofilm formation and motility. Here, we analysed the function and expression of the cdgD gene, encoding a multidomain protein that includes GGDEF-EAL domains and CHASE and PAS domains. An insertional cdgD gene mutant was constructed, and analysis of biofilm and extracellular polymeric substance production, as well as the motility phenotype indicated that cdgD encoded a functional diguanylate protein. These results were correlated with a reduced overall cellular concentration of cyclic-di-GMP in the mutant over 48 h compared with that observed in the wild-type strain, which was recovered in the complemented strain. In addition, cdgD gene expression was measured in cells growing under planktonic or biofilm conditions, and differential expression was observed when KNO3 or NH4Cl was added to the minimal medium as a nitrogen source. The transcriptional fusion of the cdgD promoter with the gene encoding the autofluorescent mCherry protein indicated that the cdgD gene was expressed both under abiotic conditions and in association with wheat roots. Reduced colonization of wheat roots was observed for the mutant compared with the wild-type strain grown in the same soil conditions. The Azospirillum-plant association begins with the motility of the bacterium towards the plant rhizosphere followed by the adsorption and adherence of these bacteria to plant roots. Therefore, it is important to study the genes that contribute to this initial interaction of the bacterium with its host plant.

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Homology-Dependent Silencing of the SC3 Gene in Schizophyllum commune

Genetics ◽

10.1093/genetics/147.2.589 ◽

1997 ◽

Vol 147 (2) ◽

pp. 589-596 ◽

Cited By ~ 1

Author(s):

Theo A Schuurs ◽

Eveline A M Schaeffer ◽

Joseph G H Wessels

Keyword(s):

Gene Silencing ◽

Genomic Dna ◽

Type Strain ◽

Cytosine Methylation ◽

Wild Type Strain ◽

Schizophyllum Commune ◽

Southern Analysis ◽

Transcriptional Level ◽

Wild Type

After introduction of extra copies of the SC3 hydrophobin gene into a wild-type strain of Schizophyllum commune, gene silencing was observed acting on both endogenous and introduced SC3 genes in primary vegetative transformants. Nuclear run-on experiments indicated that silencing acted at the transcriptional level. Southern analysis revealed that cytosine methylation of genomic DNA occurred. Moreover, SC3 silencing was suppressed by exposure to 5-azacytidine during growth. After growth of SC3-suppressed colonies from homogenized mycelium or from colonies stored at 4°, SC3 transcription was restored. However, after prolonged growth SC3 silencing was again observed. Introduction of a promoterless SC3 fragment into wild type gave less SC3 silencing.

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