Deficiency of RgpG causes major defects in cell division and biofilm formation, and deficiency of LytR-CpsAPsr family proteins leads to accumulation of cell wall antigens in culture medium by Streptococcus mutans

ABSTRACT Streptococcus mutans is known to possess rhamnose-glucose polysaccharide (RGP), a major cell wall antigen. S. mutans strains deficient in rgpG , encoding the first enzyme of the RGP biosynthesis pathway, were constructed by allelic exchange. The rgpG deficiency had no effect on growth rate but caused major defects in cell division and altered cell morphology. Unlike the coccoid wild type, the rgpG mutant existed primarily in chains of swollen, “squarish” dividing cells. Deficiency of rgpG also causes significant reduction in biofilm formation ( P &lt; 0.01). Double and triple mutants with deficiency in brpA and/or psr , genes coding for the LytR-CpsA-Psr family proteins BrpA and Psr, which were previously shown to play important roles in cell envelope biogenesis, were constructed using the rgpG mutant. There were no major differences in growth rates between the wild-type strain and the rgpG brpA and rgpG psr double mutants, but the growth rate of the rgpG brpA psr triple mutant was reduced drastically ( P &lt; 0.001). Under transmission electron microscopy, both double mutants resembled the rgpG mutant, while the triple mutant existed as giant cells with multiple asymmetric septa. When analyzed by immunoblotting, the rgpG mutant displayed major reductions in cell wall antigens compared to the wild type, while little or no signal was detected with the double and triple mutants and the brpA and psr single mutants. These results suggest that RgpG in S. mutans plays a critical role in cell division and biofilm formation and that BrpA and Psr may be responsible for attachment of cell wall antigens to the cell envelope. IMPORTANCE Streptococcus mutans , a major etiological agent of human dental caries, produces rhamnose-glucose polysaccharide (RGP) as the major cell wall antigen. This study provides direct evidence that deficiency of RgpG, the first enzyme of the RGP biosynthesis pathway, caused major defects in cell division and morphology and reduced biofilm formation by S. mutans , indicative of a significant role of RGP in cell division and biofilm formation in S. mutans . These results are novel not only in S. mutans , but also other streptococci that produce RGP. This study also shows that the LytR-CpsA-Psr family proteins BrpA and Psr in S. mutans are involved in attachment of RGP and probably other cell wall glycopolymers to the peptidoglycan. In addition, the results also suggest that BrpA and Psr may play a direct role in cell division and biofilm formation in S. mutans . This study reveals new potential targets to develop anticaries therapeutics. </jats:p

Inactivation of the fliY gene encoding a flagellar motor switch protein attenuates mobility and virulence of Leptospira interrogans strain Lai

<p>Abstract</p> <p>Background</p> <p>Pathogenic <it>Leptospira </it>species cause leptospirosis, a zoonotic disease of global importance. The spirochete displays active rotative mobility which may contribute to invasion and diffusion of the pathogen in hosts. FliY is a flagellar motor switch protein that controls flagellar motor direction in other microbes, but its role in <it>Leptospira</it>, and paricularly in pathogenicity remains unknown.</p> <p>Results</p> <p>A suicide plasmid for the <it>fliY </it>gene of <it>Leptospira interrogans </it>serogroup Icterohaemorrhagiae serovar Lai strain Lai that was disrupted by inserting the ampicillin resistance gene (<it>bla</it>) was constructed, and the inactivation of <it>fliY </it>gene in a mutant (<it>fliY</it>^-) was confirmed by PCR and Western Blot analysis. The inactivation resulted in the mRNA absence of <it>fliP </it>and <it>fliQ </it>genes which are located downstream of the <it>fliY </it>gene in the same operon. The mutant displayed visibly weakened rotative motion in liquid medium and its migration on semisolid medium was also markedly attenuated compared to the wild-type strain. Compared to the wild-type strain, the mutant showed much lower levels of adhesion to murine macrophages and apoptosis-inducing ability, and its lethality to guinea pigs was also significantly decreased.</p> <p>Conclusion</p> <p>Inactivation of <it>fliY</it>, by the method used in this paper, clearly had polar effects on downstream genes. The phentotypes observed, including lower pathogenicity, could be a consequence of <it>fliY </it>inactivation, but also a consequence of the polar effects.</p

The Role of Metagenomic Next-Generation Sequencing as a Promising Technology for Diagnosing HIV-TB Coinfection

The human immunodeficiency virus (HIV) pandemic has caused a resurgence of tuberculosis (TB), thus increasing morbidity and mortality. Moreover, HIV-TB coinfection leads to difficulties in diagnosis. Sputum smear microscopy, mycobacterial culture and GeneXpert MTB/RIF assays are generally endorsed to detect Mycobacterium tuberculosis ( M. tuberculosis ) in HIV-TB coinfection. However, these methods cannot diagnose TB in an accurate and timely manner, thus increasing the rates of HIV-associated morbidity and mortality in patients with TB. Hence, a considerable need exists for better diagnostic tools for patients with HIV-TB coinfection. Metagenomic next-generation sequencing (mNGS) is a novel detection platform widely used to assess infectious disease, antimicrobial resistance, the microbiome and human host gene expression. Herein, we summarize the advantages of mNGS for infectious disease diagnostics. We then assess the efficiency of mNGS in the detection of M. tuberculosis in different specimens and several cases of HIV-TB coinfection. We conclude that mNGS is an acceptable diagnostic method for HIV-TB coinfection, although limited research is available

The Exocyst Component Sec3 Controls Egg Chamber Development Through Notch During Drosophila Oogenesis

The exocyst complex plays multiple roles via tethering secretory or recycling vesicles to the plasma membrane. Previous studies have demonstrated that the exocyst contains eight components, which possibly have some redundant but distinct functions. It is therefore interesting to investigate the biological function of each component. Here, we found that Sec3, one component of exocyst complex, is involved in Drosophila egg chamber development. Loss of sec3 results in egg chamber fusion through the abolishment of cell differentiation. In addition, loss of sec3 increases cell numbers but decreases cell size. These defects phenocopy Notch pathway inactivation. In line with this, loss of sec3 indeed leads to Notch protein accumulation, suggesting that the loss of Sec3 inhibits the delivery of Notch onto the plasma membrane and accumulates inactive Notch in the cytoplasm. Loss of sec3 also leads to the ectopic expression of two Notch pathway target genes, Cut and FasciclinIII, which should normally be downregulated by Notch. Altogether, our study revealed that Sec3 governs egg chamber development through the regulation of Notch, and provides fresh insights into the regulation of oogenesis

Hybrid weakness and continuous flowering caused by compound expression of FTLs in Chrysanthemum morifolium × Leucanthemum paludosum intergeneric hybridization

Hybridization is an important evolutionary mechanism ubiquitous to plants. Previous studies have shown that hybrid polyploidization of cultivated chrysanthemum, ‘Zhongshanzigui’, and Leucanthemum paludosum exhibit spring-flowering traits. This study explores the function of the LpFTLs gene via the phenotype of A. thaliana after heterologous transformation of the LpFTLs gene, and analyzes the mechanism ofthe continuous flowering phenotype and heterosis of hybrid offspring. The results suggest that the flowering phenotype of hybrid offspring in spring may be related to the expression of the LpFTLs gene. Ectopic expression of Leucanthemum paludosumLpFTLs in Arabidopsis thaliana resulted in earlier flowering, indicating that the LpFTLs gene also affects the flowering time in L. paludosum. Compound expression of FTLs in C. morifolium × L. paludosum intergeneric hybridization directly leads to serious heterosis in the hybrid offspring. Moreover, continuous flowering appears to be accompanied by hybrid weakness under the balance of vegetative and reproductive growth. Therefore, in future studies on chrysanthemum breeding, a suitable balance point must be established to ensure the target flowering time under normal growth

PBP1a-deficiency causes major defects in cell division, growth and biofilm formation by Streptococcus mutans.

Streptococcus mutans, a key etiological agent of human dental caries, lives almost exclusively on the tooth surface in plaque biofilms and is known for its ability to survive and respond to various environmental insults, including low pH, and antimicrobial agents from other microbes and oral care products. In this study, a penicillin-binding protein (PBP1a)-deficient mutant, strain JB467, was generated by allelic replacement mutagenesis and analyzed for the effects of such a deficiency on S. mutans' stress tolerance response and biofilm formation. Our results so far have shown that PBP1a-deficiency in S. mutans affects growth of the deficient mutant, especially at acidic and alkaline pHs. As compared to the wild-type, UA159, the PBP1a-deficient mutant, JB467, had a reduced growth rate at pH 6.2 and did not grow at all at pH 8.2. Unlike the wild-type, the inclusion of paraquat in growth medium, especially at 2 mM or above, significantly reduced the growth rate of the mutant. Acid killing assays showed that the mutant was 15-fold more sensitive to pH 2.8 than the wild-type after 30 minutes. In a hydrogen peroxide killing assay, the mutant was 16-fold more susceptible to hydrogen peroxide (0.2%, w/v) after 90 minutes than the wild-type. Relative to the wild-type, the mutant also had an aberrant autolysis rate, indicative of compromises in cell envelope integrity. As analyzed using on 96-well plate model and spectrophotometry, biofilm formation by the mutant was decreased significantly, as compared to the wild-type. Consistently, Field Emission-SEM analysis also showed that the PBP1a-deficient mutant had limited capacity to form biofilms. TEM analysis showed that PBP1a mutant existed primarily in long rod-like cells and cells with multiple septa, as compared to the coccal wild-type. The results presented here highlight the importance of pbp1a in cell morphology, stress tolerance, and biofilm formation in S. mutans

Acid (A) and hydrogen peroxide (B) killing assays.

<p><i>S</i>. <i>mutans</i> wild-type (UA159, squares), PBP1a mutant (JB467, circles), and the complement strain (JB467C, triangles) were grown in BHI until mid-exponential phase (OD₆₀₀nm≅0.3), washed and then subjected to acid and hydrogen peroxide killing assays by incubating the cells in buffer of pH 2.8 and buffer containing 0.2% (w/v) hydrogen peroxide, respectively, for periods of time as indicated. Results showed that PBP1a deficiency in JB467 weakens the ability of the deficient mutant to tolerate low pH (A) and hydrogen peroxide (B), when compared to the wild-type. Data presented here are representatives of three independent experiments. Significant difference is indicated by * and # at <i>P</i><0.001 and <i>P</i><0.01, respectively.</p

TEM analysis.

<p><i>S</i>. <i>mutans</i> strains wild-type (UA159), PBP1a-deficient mutant (JB467), and the complement strain (JB467C) were grown in BHI, pH 7.4 to mid-exponential phase (OD₆₀₀nm≈0.3). Panel A shows differences in cell morphology with JB467 displaying elongated cells, defects in cell separation and cells with multiple septa. Images were taken at 20,000x (20k) and 50,000x (50k). Panel B highlights JB467 with a thinner layer of peptidoglycan (indicated by arrows), when compared to the wild-type and the complement strain.</p

Growth study under different pHs.

<p><i>S</i>. <i>mutans</i> wild-type (UA159, solid squares), PBP1a-deficient mutant (JB467, open circles), and the complement strain (JB467C, solid circles) were grown in BHI adjusted to pH 7.2 (A), pH 6.2 (B), and pH 8.5 (C) in Bioscreen C with sterile mineral oil overlay, and the culture optical densities were monitored every 30 minutes at 600nm. The results suggest that PBP1a-deficeincy affects growth rate and cell yield in optical density at 600 nm, especially in BHI adjusted to higher pH. The data presented here are representatives of three independent experiments.</p

Growth study in the presence of paraquat.

<p><i>S</i>. <i>mutans</i> wild-type (UA159, squares), PBP1a mutant (JB467, open circles), and the complement strain (JB467C, triangles) in the presence of increasing amounts of paraquat, and growth was continuously monitored using Bioscreen C with sterile mineral oil overlay. The data presented here are representative of three independent experiments, showing the increased susceptibility of the PBP1a-deficient strain, JB467, to 0.1 mM (A) and 5 mM (B) of paraquat (<i>P</i><0.001, as indicated by *).</p