Lactobacilli Inactivate Chlamydia trachomatis through Lactic Acid but Not H2O2

Lactobacillus species dominate the microbiome in the lower genital tract of most reproductive-age women. Producing lactic acid and H₂O₂, lactobacilli are believed to play an important role in prevention of colonization by and growth of pathogens. However, to date, there have been no reported studies characterizing how lactobacilli interact with Chlamydia trachomatis, a leading sexually transmitted bacterium. In this report, we demonstrate inactivation of C. trachomatis infectivity by culture media conditioned by Lactobacillus crispatus, L. gasseri and L. jensenii, known to be dominating organisms in the human vaginal microbiome. Lactobacillus still cultures produced lactic acid, leading to time- and concentration-dependent killing of C. trachomatis. Neutralization of the acidic media completely reversed chlamydia killing. Addition of lactic acid into Lactobacillus-unconditioned growth medium recapitulated the chlamydiacidal activity of conditioned media. The H₂O₂ concentrations in the still cultures were found to be comparable to those reported for the cervicovaginal fluid, but insufficient to inactivate chlamydiae. Aeration of Lactobacillus cultures by shaking markedly induced H₂O₂ production, but strongly inhibited Lactobacillus growth and lactic acid production, and thus severely affected acidification, leading to significantly reduced chlamydiacidal efficiency. These observations indicate lactobacilli inactivate chlamydiae primarily through maintaining acidity in a relatively hypoxic environment in the vaginal lumen with limited H₂O₂, which is consistent with the notion that women with higher vaginal pH are more prone to sexually transmitted C. trachomatis infection. In addition to lactic acid, formic acid and acetic acid also exhibited potent chlamydiacidal activities. Taken together, our findings imply that lowering the vaginal pH through engineering of the vaginal microbiome and other means will make women less susceptible to C. trachomatis infection.     

The core genome evolution of Lactobacillus crispatus as a driving force for niche competition in the human vaginal tract

The lower female reproductive tract is notoriously dominated by Lactobacillus species, among which Lactobacillus crispatus emerges for its protective and health-promoting activities. Although previous comparative genome analyses highlighted genetic and phenotypic diversity within the L. crispatus species, most studies have focused on the presence/absence of accessory genes. Here, we investigated the variation at the single nucleotide level within protein-encoding genes shared across a human-derived L. crispatus strain selection, which includes 200 currently available human-derived L. crispatus genomes as well as 41 chromosome sequences of such taxon that have been decoded in the framework of this study. Such data clearly pointed out the presence of intra-species micro-diversities that could have evolutionary significance contributing to phenotypical diversification by affecting protein domains. Specifically, two single nucleotide variations in the type II pullulanase gene sequence led to specific amino acid substitutions, possibly explaining the substantial differences in the growth performances and competition abilities observed in a multi-strain bioreactor culture simulating the vaginal environment. Accordingly, L. crispatus strains display different growth performances, suggesting that the colonisation and stable persistence in the female reproductive tract between the members of this taxon is highly variable.     

Interleukin-17F attenuates H2O2-induced cell cycle arrest

Interleukin IL-17F was expressed in colon epithelial cells and showed multiple functions in colon tumorigenesis. However, the role of IL-17F in colon cancer cell cycle progression remains unclear. In this study, we analyzed the effects of IL-17F on oxidant-induced cell cycle shift in human colon cancer cells. IL-17F overexpressing and wildtype HCT116 cells were challenged with H₂O₂. Cell cycle distribution analysis showed IL-17F attenuated H₂O₂-induced G2/M phase arrest by inhibiting S to G2/M transition. We further checked expression levels of two critical cell cycle regulators p21 and p27. The results showed that IL-17F could inhibit H₂O₂ induced p27 up-regulation. Meanwhile, IL-17F could increase the phosphorylation of p38 after H₂O₂ treatment. The regulations of p27 level and p38 activity may contribute to the impaired G2/M phase arrest by IL-17F. Taken together, our findings extend IL-17F as an important factor in colon cancer development and provide new insight into the signaling pathway.     

Oxidative stress induces alkaloid production in Uncaria tomentosa root and cell cultures in bioreactors

The effect of oxidative stress on indole alkaloids accumulation by cell suspensions and root cultures of Uncaria tomentosa in bioreactors was investigated. Hydrogen peroxide (H₂O₂, 200 μM) added to U. tomentosa cell suspension cultures in shaken flasks induced the production of monoterpenoid oxindole alkaloids (MOA) up to 40.0 μg/L. In a stirred tank bioreactor, MOA were enhanced by exogenous H₂O₂ (200 μM) from no detection up to 59.3 μg/L. Root cultures grew linearly in shaken flasks with a μ=0.045 days^?1 and maximum biomass of 12.08±1.24 g DW/L (at day 30). Roots accumulated 3α‐dihydrocadambine (DHC) 2354.3±244.8 μg/g DW (at day 40) and MOA 348.2±32.1 μg/g DW (at day 18). Exogenous addition of H₂O₂ had a differential effect on DHC and MOA production in shaken flasks. At 200 μM H₂O₂, MOA were enhanced by 56% and DHC by 30%; while addition of 800 and 1000 μM H₂O₂, reduced by 30–40% DHC accumulation without change in MOA. Root cultures in the airlift reactor produced extracellular H₂O₂ with a characteristic biphasic profile after changing aeration. Maximum MOA was 9.06 mg/L at day 60 while at this time roots reached ca. 1 mg/L of DHC. Intracellular H₂O₂ in root cultures growing in the bioreactor was 0.87 μmol/g DW compared to 0.26 μmol/g DW of shaken flasks cultures. These results were in agreement with a higher activity of the antioxidant enzymes superoxide dismutase and peroxidase by 6‐ and 2‐times, respectively. U. tomentosa roots growing in the airlift bioreactor were exposed to an oxidative stress and their antioxidant system was active allowing them to produce oxindole alkaloids.     

Probiotic Properties of Lactobacillus crispatus 2,029: Homeostatic Interaction with Cervicovaginal Epithelial Cells and Antagonistic Activity to Genitourinary Pathogens

Lactobacillus crispatus 2029 isolated upon investigation of vaginal lactobacilli of healthy women of reproductive age was selected as a probiotic candidate. The aim of the present study was elucidation of the role of L. crispatus 2029 in resistance of the female reproductive tract to genitourinary pathogens using cervicovaginal epithelial model. Lactobacillus crispatus 2029 has surface layers (S-layers), which completely surround cells as the outermost component of their envelope. S-layers are responsible for the adhesion of lactobacilli on the surface of cervicovaginal epithelial cells. Study of interactions between L. crispatus 2029 and a type IV collagen, a major molecular component of epithelial cell extracellular matrix, showed that 125I-labeled type IV collagen binds to lactobacilli with high affinity (Kd = (8.0 ± 0.7) × 10^?10 M). Lactobacillus crispatus 2029 consistently colonized epithelial cells. There were no toxicity, epithelial damage and apoptosis after 24 h of colonization. Electronic microscope images demonstrated intimate association between L. crispatus 2029 and epithelial cells. Upon binding to epithelial cells, lactobacilli were recognized by toll-like 2/6 receptors. Lactobacillus crispatus induced NF-κB activation in epithelial cells and did not induce expression of innate immunity mediators IL-8, IL-1β, IL-1α and TNF-α. Lactobacillus crispatus 2029 inhibited IL-8 production in epithelial cells induced by MALP-2 and increased production of anti-inflammatory cytokine IL-6, maintaining the homeostasis of female reproductive tract. Lactobacillus crispatus 2029 produced H₂O₂ and provided wide spectrum of antagonistic activity increasing colonization resistance to urinary tract infections by bacterial vaginosis and vulvovaginal candidiasis associated agents.     

人生殖道源乳酸杆菌的筛选及在小鼠阴道假丝酵母病模型中的应用评价

【目的】分离筛选人阴道环境中具有益生特性的乳酸杆菌,探索外阴阴道假丝酵母病的益生菌疗法。【方法】利用含1%碳酸钙的de Man,Rogosa and Sharpe (MRS)培养基从无症状育龄女性阴道分泌物中分离乳酸杆菌,采用共培养方法评价其对白色念珠菌(Candida albicans)的抑制作用,通过对乳酸杆菌的耐酸性能、体外聚集特性和黏附能力测试考察其益生特性,并进行乳酸杆菌株功能化组合。通过构建小鼠外阴阴道假丝酵母病模型,初步探索乳酸杆菌株组合对C. albicans的抑制作用。【结果】从53个样品中分离得到19株乳酸杆菌,筛选获得4株乳酸杆菌(Lactobacillus crispatus ZH08、L. fermentumZH09、L. fermentum ZH11和L. crispatus ZH17)具有较强抑制C. albicans生长的能力。4株乳酸杆菌均能耐受低pH环境,能快速降低培养液pH。其中2株L. fermentum具有更强的抑制活性,能在24 h内快速抑制C. albicans生长,抑制率可达到95%以上;另2株L. crispatus具有更强的聚集特性和...     

Characterization of vaginal lactobacilli in women after kidney transplantation

Limited number of publications described vaginal microflora after kidney transplantation. Our PubMed search revealed only 18 publications including words “vaginal bacteria &; kidney transplant” in the period of 1978–2011. The aim of this study was to characterize lactobacilli isolated from vaginal swabs of women after kidney transplantation, compared with healthy women. Eighteen renal transplant recipients (mean age 36.1) and 20 healthy women (mean age 36.0) were evaluated. Lactobacilli were cultured on MRS and Columbia blood agars. Biochemical identification with API 50 CHL (bioMerieux, Marcy L’Etoile, France) and multiplex PCR according to Song et al. was performed. Lactobacilli were tested for production of H₂O₂. Minimal inhibitory concentrations (MICs) of selected antimicrobial agents were determined with E-tests (bioMerieux, Marcy L’Etoile, France) and interpreted with CLSI and EUCAST criteria. No bacterial vaginosis was found among studied women. Two strains of group I were identified as Lactobacillus delbrueckii; 18 strains as Lactobacillus gasseri and 15 strains as Lactobacillus crispatus. Only 3 strains from group II were not identified by species-specific mPCR. Group IV was represented with 2 unidentified strains. Vaginal lactobacilli isolated from healthy women represented more homogenous group compared with heterogenous renal transplant recipients. Biochemical identification of lactobacilli by API 50 CHL kits was concordant with mPCR results only in 7 cases (17.5%), all 7 strains were identified as L. crispatus. Majority (93%) of lactobacilli were H₂O₂ producers. All isolated lactobacilli (100%) demonstrated high resistance to metronidazole (MIC > 256 μg/ml). Only 2 strains resistant to vancomycin (MICs: 32 and 256 μg/ml respectively), in the study and control group, and one to moxifloxacin (MIC = 32 μg/ml), were found. Resistance to metronidazole and vancomycin was concordant in CLSI and EUCAST (2010) criteria. Although significant differences between lactobacilli isolated from vaginas of kidney transplant and healthy women were not demonstrated, we demonstrated strains resistant to metronidazole, vancomycin and moxifloxacin in groups of examined women. Our study was performed on a small group of kidney transplant recipients and further more detailed molecular studies on a larger group of patients are required to confirm our results.     

山黧豆根系对H2O2诱导氧化胁迫的生理应答

以水培7d苗龄的山黧豆幼苗为材料,向水培溶液中施加不同浓度H2O2处理山黧豆幼苗24h,分析山黧豆根系受氧化胁迫的程度与抗氧化系统的应答特征,以揭示山黧豆对氧化胁迫的耐受机制。结果显示:(1)随外源H2O2处理浓度的不断增加,山黧豆幼苗侧根的数目无显著变化,而其根的鲜重则显著降低。(2)同时,根系组织的内源H2O2染色范围和程度显著增高,但根尖区域始终保持较低水平的H2O2;相反,O-·2染色范围和程度明显减少,根尖区域却始终保持较高水平的O-·2。(3)同期根系抗坏血酸(ASC)含量及过氧化氢酶(CAT)、过氧化物酶(POD)与抗坏血酸过氧化物酶(APX)的活性均表现出了先升高后降低的趋势,而超氧化物歧化酶(SOD)一直表现为持续上升的趋势。研究表明,在外源H2O2胁迫条件下,山黧豆根系O-·2的积累可能与其生长和活力呈正相关,而根系H2O2的积累则与其受氧化胁迫程度呈正相关;低浓度的H2O2处理可以提高山黧豆抗氧化系统对体内活性氧的清除能力。     

The generation of H2O2 in the xylem of Zinnia elegans is mediated by an NADPH-oxidase-like enzyme

The nature of the enzymatic system responsible for the generation of H₂O₂ in the lignifying xylem of Zinnia elegans (L.) was studied using the starch/KI method for monitoring H₂O₂ production and the nitroblue tetrazolium method for monitoring superoxide production. The results showed that lignifying xylem tissues are able to accumulate H₂O₂ and to sustain H₂O₂ production. Hydrogen peroxide production in the xylem of Z. elegans was sensitive to pyridine, imidazole, quinacrine and diphenylene iodonium, which are inhibitors of phagocytic plasma-membrane NADPH oxidase. The sensitivity of H₂O₂ production to the inhibitor of phospholipase C, neomycin, and to the inhibitor of protein kinase, staurosporine, and its reversion by the inhibitor of protein phosphatases, cantharidin, pointed to the analogies existing between the mechanism of H₂O₂ production in lignifying xylem and the oxidative burst observed during the hypersensitive plant cell response. A further support for the participation of an NADPH-oxidase-like activity in H₂O₂ production in lignifying xylem was obtained from the observation that areas of H₂O₂ production were superimposed on areas producing superoxide anion, the suspected product of NADPH oxidase, although attempts to demonstrate the existence of superoxide dismutase activity in intercellular washing fluid from Z. elegans were unsuccessful. Even so, the levels of NADPH-oxidase-like activity in microsomal fractions, and of peroxidase in intercellular washing fluids, are consistent with a role for NADPH oxidase in the delivery of H₂O₂ which may be further used by xylem peroxidases for the synthesis of lignins. This hypothesis was further confirmed through a direct histochemical probe based on the H₂O₂-dependent oxidation of tetramethylbenzidine by xylem cell wall peroxidases. These results are the first evidence for the existence of an NADPH oxidase responsible for supplying H₂O₂ to peroxidase in the lignifying xylem of Z. elegans. Received: 6 February 1998 / Accepted: 14 August 1998     

Interindividual differences in initial DNA repair capacity when evaluating H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced DNA damage in extended-term cultures of human lymphocytes using the comet assay

It has been suggested that extended-term cultures of human lymphocytes could be used as a complement to cell lines based on transformed cells when testing the genotoxicity of chemicals. To investigate whether the pattern of induced DNA damage and its subsequent repair differs significantly between cultures based on different blood donors, hydrogen peroxide (H₂O₂)-induced DNA damage was measured in cultures from four different subjects using the comet assay. The DNA damage was significantly increased in all cultures after 10 min exposure to 0.25 mmol/L H₂O₂, and there was a significant decrease in the H₂O₂-induced DNA damage in all cultures after 30 min of DNA repair. The level of damage varied between the different donors, especially after the repair. Using PCR and DNA sequencing, exon 5 of the p53 gene was sequenced in the lymphocytes from the donors with the lowest and highest residual damage. No such mutation was found. Mouse lymphoma L5178Y cells carrying the p53 mutation in exon 5 were included as a reference. These cells were found to be less sensitive toward the H₂O₂-induced DNA damage, and they were also found to have a rather low DNA repair capacity. The demonstrated variation in H₂O₂-induced DNA damage and DNA repair capacity between the cultures from the different subjects may be important from a risk assessment perspective, but is obviously not of decisive importance when it comes to the development of a routine assay for genotoxicity.     

Involvement of manganese peroxidase in the transformation of macromolecules from low-rank coal by Phanerochaete chrysosporium

Manganese peroxidase (Mn peroxidase) catalyses the oxidation of Mn(II) to Mn(III), a diffusible non-specific oxidant likely to be involved in the transformation of polyphenolic macromolecules from brown coal by the white-rot fungus Phanerochaete chrysosporium. We report here that solubilised macromolecules from Morwell brown coal were depolymerised by Mn(III) ions when incubated under hyperbaric O₂. However, under N₂ or air they were polymerised, suggesting that net depolymerisation by Mn(III) requires molecular oxygen to inhibit coupling of coal radicals. Coal macromolecules were also polymerised when separated by a semipermeable membrane from a culture of P. chrysosporium or from a solution of Mn peroxidase, Mn(II) and H₂O₂, probably by Mn(III) crossing the membrane. In oxygenated cultures in which Mn peroxidase␣was up-regulated by Mn(II), the extent of depolymerisation correlated with cumulative Mn peroxidase activity suggesting that Mn-peroxidase-generated Mn(III) has a central role in initial depolymerisation of coal molecules in vivo. However, mutant ME446-B17-1, which produces Mn peroxidase but not lignin peroxidase, polymerised coal macromolecules in oxygenated cultures. In sum, it appears Mn peroxidase can both polymerise and depolymerise brown coal macromolecules and that, in vivo, both hyperbaric O₂ and lignin peroxidase are also required to force net depolymerisation to products assimilable by cells. Received: 4 September 1997 / Received revision: 29 January 1998 / Accepted: 30 January 1998     

Enhanced gellan gum production by hydrogen peroxide (H2O2) induced oxidative stresses in Sphingomonas paucimobilis

In this study, the effect of H₂O₂-induced oxidative stress on gellan gum production and cell growth were investigated. Gellan gum production was improved and cell growth was inhibited by H₂O₂. A multiple H₂O₂ stresses with different concentrations were developed to optimize gellan gum production. A maximal gellan gum yield (22.52 g/L), which was 35.58 % higher than the control, was observed with 2, 2, 3, 4 mmol/L H₂O₂ added at 6, 12, 18, 24 h, respectively. Moreover, UDP-glucose pyrophosphorylase activity and glucosyltransferase activity were increased with H₂O₂ stresses. This new strategy of multiple H₂O₂-induced oxidative stresses would be further applied to gellan gum production in future study.     

PLC1 and H2O2 Mediated the Allelopathic Effect of Oridonin on Root Growth in Arabidopsis thaliana

Oridonin is a diterpenoid isolated from medicinal herb Rabdosia rubescens (Hemsl.) Hara (Lamiaceae), which has an allelopathic effect on plants. Phospholipase C (PLC1) and hydrogen peroxide (H₂O₂) are involved in many biotic or abiotic stress responses. Using the 16-day-old seedlings of Arabidopsis thaliana ecotype (WT) and PLC1-deficient mutant (plc1) as materials (treated with 10 μM or 60 μM oridonin for 72 h), the effect of oridonin on root growth regulating by PLC1 and H₂O₂ was investigated. The results showed that the promoting of root growth was about 6.9% at 10 μmol L^?1 oridonin and the inhibiting of root growth was about 19.73% at 60 μmol L^?1 oridonin in WT, the inhibiting of root growth was about 10.5% and 41.2% at 10 mol L^?1 and 60 mol L^?1 oridonin, respectively, in plc1. The expression of ARR1, ARR12, and AHK3 was promoted at low concentrations of oridonin and inhibited at high concentrations in WT, whereas the expression of ARR1 and ARR12 was inhibited with the increase of oridonin concentration in plc1. This suggested that PLC1 was involved in the root growth regulation of oridonin. H₂O₂ was promoted by oridonin with concentration dependence pattern in root cells. Oridonin increased the activity of antioxidant enzymes in both WT and plc1, but the activity of antioxidant enzymes in plc1 was lower than WT. This indicated that PLC1 involved in the activation of antioxidant enzymes promoted by the oridonin. Exogenous CaCl₂ facilitated the accumulation of H₂O₂ in both WT and plc1. And the H₂O₂ of WT was obviously higher than that of plc1. The root growth of WT was inhibited by CaCl₂ with the increase of oridonin. However, there is no effect of CaCl₂ on the root growth in plc1. This reflected that PLC1 positively involved in the regulation of Ca²⁺ on the H₂O₂ and the inhibition effect of Ca²⁺ on the root growth under oridonin treatment. PA promoted the H₂O₂ and suppressed the root growth under oridonin treatment in both WT and plc1. In plc1, PA facilitated the root growth with no oridonin and inhibited the root growth with the increase of oridonin. This reflected that PLC1 positively regulated the promotion effect of PA on the root growth under high oridonin treatment. PLC1 mediated oridonin (10 and 60 mol L^?1) to regulate H₂O₂ levels in A. thaliana seedlings, thereby regulating root tip cell morphology and mitosis. These results demonstrated that PLC1 mediated the low-promotion and high-inhibition effect of oridonin on the root growth in A. thaliana by regulating the concentrations of Ca²⁺ and PA, and further affecting the intracellular H₂O₂ level.

     

High-Level Expression of Heme-Dependent Catalase Gene <Emphasis Type="Italic">katA</Emphasis> from <Emphasis Type="Italic">Lactobacillus Sakei</Emphasis> Protects <Emphasis Type="Italic">Lactobacillus Rhamnosus</Emphasis> from Oxidative Stress

Lactic acid bacteria (LAB) are generally sensitive to hydrogen peroxide (H₂O₂), Lactobacillus sakei YSI8 is one of the very few LAB strains able to degrade H₂O₂ through the action of a heme-dependent catalase. Lactobacillus rhamnosus strains are very important probiotic starter cultures in meat product fermentation, but they are deficient in catalase. In this study, the effect of heterologous expression of L. sakei catalase gene katA in L. rhamnosus on its oxidative stress resistance was tested. The recombinant L. rhamnosus AS 1.2466 was able to decompose H₂O₂ and the catalase activity reached 2.85 μmol H₂O₂/min/10⁸ c.f.u. Furthermore, the expression of the katA gene in L. rhamnosus conferred enhanced oxidative resistance on the host. The survival ratios after short-term H₂O₂ challenge were increased 600 and 10⁴-fold at exponential and stationary phase, respectively. Further, viable cells were 100-fold higher in long-term aerated cultures. Simulation experiment demonstrated that both growth and catalase activity of recombinant L. rhamnosus displayed high stability under environmental conditions similar to those encountered during sausage fermentation.     

Biosynthesis of NiFe2O4@Ag Nanocomposite and Assessment of Its Effect on Expression of norA Gene in Staphylococcus aureus

Activity of norA efflux pump has been known as a resistance mechanism to antibiotics like ciprofloxacin in Staphylococcus aureus. This study was carried out to assess the effect of biosynthesized NiFe₂O₄@Ag nanocomposite on expression of norA gene in Staphylococcus aureus. In this experimental study, 30 clinical samples were collected from patients hospitalized at different hospitals in Guilan Province, Iran. Then, clinical isolates of S. aureus were identified by standard microbiological tests. Antimicrobial susceptibility tests of clinical and standard strains of S. aureus were done by disk diffusion method according to CLSI guideline. Fourier transform infrared spectroscopy (FT‐IR) was used to analyze the various functional groups present in the biosynthesized NiFe₂O₄@Ag nanocomposite. This analysis confirmed the formation of alga proteins coated on magnetite nanocomposite. X‐ray diffraction (XRD) verified the crystalline structure of NiFe₂O₄@Ag and the deposition of silver on the surface of NiFe₂O₄. Energy dispersive X‐ray mapping (EDX‐map) analysis confirmed the existence of Ag, Ni, Fe and O in the final product. Scanning electron microscopy (SEM) confirmed that the nanocomposites were spherical in shape and Transmission electron microscopy (TEM) results revealed that the NiFe₂O₄@Ag had the particle size about 100 nm. Antibacterial activity of NiFe₂O₄@Ag alone and combined with ciprofloxacin was evaluated using the disk assay method, and minimum inhibitory concentration (MIC) by broth dilution method. Afterwards, the expression of norA efflux pump gene with and without of NiFe₂O₄@Ag nanocomposite and ciprofloxacin was evaluated by Real‐Time PCR. Real‐Time PCR results demonstrated that the expression of norA gene in the strains exposed to both NiFe₂O₄@Ag nanocomposite (1/4 MIC) and ciprofloxacin (1/8 MIC) significantly reduced in comparison to untreated strains. This study reveals that, when NiFe₂O₄@Ag nanocomposite is combined with ciprofloxacin, the inhibitory effect of ciprofloxacin increases against growth of S. aureus. Therefore, NiFe₂O₄@Ag nanocomposite can be considered as an effective factor to decrease the growth of S. aureus along with ciprofloxacin.     

Unique Insights in the Cervicovaginal Lactobacillus iners and L. crispatus Proteomes and Their Associations with Microbiota Dysbiosis

Background

A Lactobacillus-dominated cervicovaginal microbiota (VMB) protects women from adverse reproductive health outcomes, but the role of L. iners in the VMB is poorly understood. Our aim was to explore the association between the cervicovaginal L. iners and L. crispatus proteomes and VMB composition.

Methods

The vaginal proteomes of 50 Rwandan women at high HIV risk, grouped into four VMB groups (based on 16S rDNA microarray results), were investigated by mass spectrometry using cervicovaginal lavage (CVL) samples. Only samples with positive 16S results for L. iners and/or L. crispatus within each group were included in subsequent comparative protein analyses: Lactobacillus crispatus-dominated VMB cluster (with 16S-proven L. iners (n_i) = 0, and with 16S-proven L. crispatus (n_c) = 5), L. iners-dominated VMB cluster (n_i = 11, n_c = 4), moderate dysbiosis (n_i = 12, n_c = 2); and severe dysbiosis (n_i = 8, n_c = 2). The relative abundances of proteins that were considered specific for L. iners and L. crispatus were compared among VMB groups.

Results

Forty Lactobacillus proteins were identified of which 7 were specific for L. iners and 11 for L. crispatus. The relative abundances of L. iners DNA starvation/stationary phase protection protein (DPS), and the glycolysis enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glucose-6-phosphate isomerase (GPI), were significantly decreased in women with L. iners-containing dysbiosis compared to women with a L. iners-dominated VMB, independent of vaginal pH and L. iners abundance. Furthermore, L. iners DPS, GAPDH, GPI, and fructose-bisphosphate aldolase (ALDO) were significantly negatively associated with vaginal pH. Glycolysis enzymes of L. crispatus showed a similar negative, but nonsignificant, trend related to dysbiosis.

Conclusions

Most identified Lactobacillus proteins had conserved intracellular functions, but their high abundance in CVL supernatant might imply an additional extracellular (moonlighting) role. Our findings suggest that these proteins can be important in maintaining a Lactobacillus-dominated VMB. Functional studies are needed to investigate their roles in vaginal bacterial communities and whether they can be used to prevent vaginal dysbiosis.     

Exogenous application of salicylic acid alleviates cadmium toxicity and reduces hydrogen peroxide accumulation in root apoplasts of Phaseolus aureus and Vicia sativa

We examined ameliorative effects of salicylic acid (SA) on two cadmium (Cd)-stressed legume crops with different Cd tolerances, viz. Phaseolus aureus (Cd sensitive) and Vicia sativa (Cd tolerant). Cd at 50 μM significantly increased the production of hydrogen peroxide (H₂O₂) and superoxide anion (O₂^·−) in root apoplasts of P. aureus and V. sativa. When comparing the two species, we determined that Cd-induced production of H₂O₂ and O₂^·− was more pronounced in P. aureus root apoplasts than in V. sativa root apoplasts. V. sativa had higher activities of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) than P. aureus in root symplasts and apoplasts. Seed-soaking pretreatment with 100 μM SA decreased Cd-induced production of H₂O₂ and O₂^·− in apoplasts of both species, and increased activities of symplastic and apoplastic SOD, symplastic APX, and apoplastic CAT under Cd stress. Hence, SA-induced Cd tolerances in P. aureus and V. sativa are likely associated with increases in symplastic and apoplastic antioxidant enzyme activities.     

Copper-mediated oxidative burst in Nicotiana tabacum L. cv. Bright Yellow 2 cell suspension cultures

Summary.  In cell suspension cultures of Nicotiana tabacum L. cv. Bright Yellow 2 (BY-2) a rapid and concentration-dependent accumulation of H₂O₂ is induced by excess concentrations of copper (up to 100 μM). This specific and early response towards copper stress was shown to be extracellular. Addition of 300 U of catalase per ml decreased the level of H₂O₂. Superoxide dismutase (5 U/ml) induced an increase in H₂O₂ production by 22.2%. This indicates that at least part of the H₂O₂ is produced by dismutation of superoxide. Pretreatment of the cell cultures with the NAD(P)H oxidase inhibitors diphenylene iodonium (2 and 10 μM) and quinacrine (1 and 5 mM) prevented the generation of H₂O₂ under copper stress for 90%. The influence of the pH on the H₂O₂ production revealed the possible involvement of cell-wall-dependent peroxidases in the generation of reactive oxygen species after copper stress. Received May 20, 2002; accepted July 26, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Plant Physiology, Department of Biology, University of Antwerp (RUCA), Groenenborgerlaan 171, 2020 Antwerp, Belgium.     

An efflux inhibitor of the MacAB pump in Salmonella enterica serovar Typhimurium

Multidrug efflux pumps play an important role in bacterial multidrug resistance by actively excreting antibiotics. The ATP-binding cassette–type drug efflux pump MacAB was originally reported as a macrolide-specific pump. MacAB is also known to be required for the virulence of Salmonella enterica serovar Typhimurium following oral infection in mice. Here, we performed a screening of inhibitors of Salmonella MacAB and found a compound that increased the susceptibility of a MacAB-expressing strain to macrolides. It was previously reported that MacAB is required to resist peroxide-mediated killing in vitro and that a supernatant of wild-type Salmonella rescues the growth defect of a macAB mutant in H₂O₂. In this study, we also found that the MacAB inhibitor reduced the ability of the supernatant to rescue Salmonella cells in H₂O₂. This compound could lead to a better understanding of the function of MacAB.     

Induction of alkalinization and an oxidative burst by low doses of cycloheximide in soybean cells

Soybean (Glycine max L. Merr.) Cell-suspension cultures inoculated with avirulent Pseudomonas syringae pv. glycinea bacteria generated a sustained oxidative burst 3–6 h after the infection. The H₂O₂ production was not dependent on protein biosynthesis but, surprisingly, cycloheximide itself was a very strong inducer of the oxidative burst and of the alkalinization measured in the cell culture medium. Both responses were activated in a very similar manner by inhibitors of protein phosphatases, implicating a phosphorylation change evoked by cycloheximide as a trigger for the elicitation. The activation of the oxidative burst was totally blocked by the kinase inhibitor K252a. The alkalinization response preceded the oxidative burst. The generation of H₂O₂ depleted the medium of H⁺ but the expected alkalinization of about one pH-unit did not occur. The H₂O₂ production by the plasma membrane oxidase must therefore be charge-compensated, likely via H⁺-channel activity. Received: 4 October 1997 / Accepted: 12 May 1998