Resuscitation-promoting Factors Research Articles

Insight in lag phase of Listeria monocytogenes during enrichment through proteomic and transcriptomic responses

The dynamics of the enrichment-based detection procedure of the foodborne pathogen Listeria monocytogenes from food still remains poorly understood. This enrichment is crucial in the reliable detection of this pathogen and more insight into the recovery mechanism during this step is important to advance our understanding of lag phase behaviour during enrichment. In this study we combined transcriptomic and proteomic analyses to better understand the physiological processes within the lag phase of L. monocytogenes during enrichment. Upon transfer of BHI-cultured stationary phase L. monocytogenes cells to half-Fraser enrichment broth (HFB), motility-associated genes and proteins were downregulated, while expression of metal uptake transporters, resuscitation-promoting factors that stimulate growth from dormancy, antibiotic efflux pumps and oxidative stress proteins were upregulated. Next to this, when cells with a heat stress history were cultured in enrichment broth, proteins necessary for recovery were upregulated with functions in DNA-damage repair, protein refolding, cell-wall repair, and zinc transport. Proteomic results pointed to possible factors that support shortening the lag duration, including the addition of 10 µM zinc and the addition of spent HFB containing presumed concentrations of resuscitation-promoting factors. However, these interventions did not lead to biologically relevant reduction of lag phase. Also, when cells were enriched in spent HFB, final cell concentrations were similar to enrichments in fresh HFB, indicating that the enrichment broth seems not to lack critical substrates. Concludingly, this study gives insight into the proteomic changes in the lag phase during enrichment and shows that supplementation of HFB is not the best strategy to optimize the current enrichment method.

Plasma-generated nitric oxide water: A promising strategy to combat bacterial dormancy (VBNC state) in environmental contaminant Micrococcus luteus

The viable but non-culturable (VBNC) is an inactive state, and certain bacteria can enter under adverse conditions. The VBNC state challenges the environment, food safety, and public health since VBNCs may resuscitate and pose a risk to human health. The aim of this study was to investigate the effect of plasma-generated nitric oxide water (PG-NOW) on airborne contaminant Micrococcus luteus (M. luteus) and examine its potential to induce the VBNC state. The essential conditions for bacteria to enter VBNC state are low metabolic activity and rare or no culturable counts. The results indicated that PG-NOW effectively eliminates M. luteus, and the remaining bacteria are in culturable condition. Moreover, the conventional cultured-based method combined with a propidium iodide monoazide quantitative PCR (PMAxxTM-qPCR) showed no significant VBNC induction and moderate culturable counts. Results from the qPCR revealed that gene levels in PG-NOW treated bacteria related to resuscitation-promoting factors, amino acid biosynthesis, and fatty acid metabolism were notably upregulated. PG-NOW inactivated M. luteus showed negligible VBNC formation and alleviated infection ability in lung cells. This study provides new insights into the potential use of PG-NOW reactive species for the prevention and control of the VBNC state of M. luteus.

The resuscitation-promoting factor (Rpf) from Micrococcus luteus and its putative reaction product 1,6-anhydro-MurNAc increase culturability of environmental bacteria.

Dormant bacterial cells do not divide and are not immediately culturable, but they persist in a state of low metabolic activity, a physiological state having clinical relevance, for instance in latent tuberculosis. Resuscitation-promoting factors (Rpfs) are proteins that act as signalling molecules mediating growth and replication. In this study we aimed to test the effect of Rpfs from Micrococcus luteus on the number and diversity of cultured bacteria using insect and soil samples, and to examine if the increase in culturability could be reproduced with the putative reaction product of Rpf, 1,6-anhydro-N-acetylmuramic acid (1,6-anhydro-MurNAc). The rpf gene from Micrococcus luteus was amplified and cloned into a pET21b expression vector and the protein was expressed in Escherichia coli BL21(DE3) cells and purified by affinity chromatography using a hexa-histidine tag. 1,6-Anhydro-MurNAc was prepared using reported chemical synthesis methods. Recombinant Rpf protein or 1,6-anhydro-MurNAc were added to R2A cultivation media, and their effect on the culturability of bacteria from eight environmental samples including four cockroach guts and four soils was examined. Colony-forming units, 16S rRNA gene copies and Illumina amplicon sequencing of the 16S rRNA gene were measured for all eight samples subjected to three different treatments: Rpf, 1,6-anhydro-MurNAc or blank control. Both Rpf and 1,6-anhydro-MurNAc increased the number of colony-forming units and of 16S rRNA gene copies across the samples although the protein was more effective. The Rpf and 1,6-anhydro-MurNAc promoted the cultivation of a diverse set of bacteria and in particular certain clades of the phyla Actinomycetota and Bacillota . This study opens the path for improved cultivation strategies aiming to isolate and study yet undescribed living bacterial organisms.

Repurposing of antibacterial compounds for suppression of Mycobacterium tuberculosis dormancy reactivation by targeting resuscitation-promoting factors B

Tuberculosis infection has always been a global concern for public health, and the mortality rate has increased tremendously every year. The ability of the resuscitation Mycobacterium tuberculosis (Mtb) from the dormant state is one of the major reasons for the epidemic spread of tuberculosis infection, especially latent tuberculosis infection (LTBI). The element that encourages resuscitation, RpfB (resuscitation-promoting factors B), is mostly in charge of bringing Mtb out of slumber. This reason makes RpfB a promising target for developing tuberculosis drugs because of the effects of latent tuberculosis. Therefore, this work was executed using a computational three-level screening of the Selleckhem antibiotics database consisting of 462 antibiotics against the ligand binding region of the RpfB protein, followed by an estimation of binding free energy for ideal identification and confirmation of potential RpfB inhibitor. Subsequently, three antibiotic drug molecules, i.e., Amikacin hydrate (−66.87 kcal/mol), Isepamicin sulphate (−60.8 kcal/mol), and Bekanamycin (−46.89 kcal/mol), were selected on the basis of their binding free energy value for further computational studies in comparison to reference ligand, 4-benzoyl-2-nitrophenyl thiocyanate (NPT7). Based on the intermolecular interaction profiling, 200 ns molecular dynamic simulation (MD), post-simulation analysis and principal component analysis (PCA), the selected antibiotics showed substantial stability with the RpfB protein compared to the NPT7 inhibitor. Conclusively based on the computational results, the preferred drugs can be potent inhibitors of the RpfB protein, which can be further validated using in vivo research and in vitro enzyme inhibition to understand their therapeutic activity against tuberculosis infection. Communicated by Ramaswamy H. Sarma

Time to detection of Mycobacterium tuberculosis using culture filtrate H37rv supplementation on MGIT 960 System

Background: The fundamental priority program for controlling Tuberculosis (TB) is accurate and rapid diagnosis. Variable Mycobacterium tuberculosis (MTB) phenotypes were discovered in sputum samples from pulmonary TB (PTB) patients. Active replicating bacteria and differentially culturable tubercle bacteria (DCTB) are present in the sputum. Differentially culturable tubercle bacilli did not grow on the standard medium. Culture filtrate (CF) H37Rv supplementation containing resuscitation-promoting factors (Rpfs) has the potential to increase sensitivity and reduce time to detection (TTD) on culture-based diagnostics. Methods: This study was a true experimental laboratory. A clinical sample was taken from the sputum of 15 PTB patients, which was "MTB detected" by Xpert MTB/RIF assay. Sputum samples were divided into two groups and inoculated on MGIT 960 in one group with CF-H37Rv supplementation and without in the other as standard MGIT medium. Results: On both standard MGIT medium and MGIT+CF H37Rv, the recovery rate was 100%. Seven samples (47%) show a growth promotion effect with CF H37Rv supplementation (ΔTTD > 10 hours), while eight samples (53%) show no change in TTD (ΔTTD < 10 hours). There is no evidence that CF H37Rv supplementation inhibits growth. Six of the seven samples with ΔTTD > 10 jam (85,7%) were dominated by samples with acid-fast bacilli (AFB) grading 1+, which could be an assumption related to re-treatment PTB cases, chronic cases, treatment stages, TB-HIV, or follow up of treatments. Conclusion: There was no difference in recovery rate or TTD between standard MGIT medium and MGIT+CF H37Rv. The supplementation of culture filtrate H37Rv has a greater advantage on samples with low bacterial load (AFB 1+).

Evidence, Challenges, and Knowledge Gaps Regarding Latent Tuberculosis in Animals.

Mycobacterium bovis and other Mycobacterium tuberculosis complex (MTBC) pathogens that cause domestic animal and wildlife tuberculosis have received considerably less attention than M. tuberculosis, the primary cause of human tuberculosis (TB). Human TB studies have shown that different stages of infection can exist, driven by host–pathogen interactions. This results in the emergence of heterogeneous subpopulations of mycobacteria in different phenotypic states, which range from actively replicating (AR) cells to viable but slowly or non-replicating (VBNR), viable but non-culturable (VBNC), and dormant mycobacteria. The VBNR, VBNC, and dormant subpopulations are believed to underlie latent tuberculosis (LTB) in humans; however, it is unclear if a similar phenomenon could be happening in animals. This review discusses the evidence, challenges, and knowledge gaps regarding LTB in animals, and possible host–pathogen differences in the MTBC strains M. tuberculosis and M. bovis during infection. We further consider models that might be adapted from human TB research to investigate how the different phenotypic states of bacteria could influence TB stages in animals. In addition, we explore potential host biomarkers and mycobacterial changes in the DosR regulon, transcriptional sigma factors, and resuscitation-promoting factors that may influence the development of LTB.

复苏促进因子辅助单细胞分离筛选青藏高原低温联苯降解潜力菌

<p indent="0mm">The mining of microbial resources has always been the focus of researchers. In the traditional microbial isolation method, due to the competition among dominant bacteria and the limitation of culture conditions, many microorganisms are unculturable, which are referred to as “microbial dark matter”. Isolating bacteria before they begin to grow can eliminate interspecific competition and provide a conductive environment for subsequent cultivation. However, this approach does not alleviate the growth limitations caused by laboratory culture conditions on many microorganisms. Resuscitation-promoting factors can resuscitate and promote the growth of many bacteria. It has been widely used by researchers to isolate functional strains. Therefore, addition of the resuscitation-promoting factor can improve the isolation efficiency of low-activity and slow-growing microorganisms. However, no study has investigated the performance of the combination of these two methods. The Qinghai-Tibet Plateau harbors a huge reserve of microbial resources. The recent industrial development has accelerated resource exploitation in cold area in recent years. The fragile ecological environment of the Qinghai-Tibet Plateau is threatened by several factors including the crude oil and persistent organic pollutants. Given the prolonged low temperature environment in the plateau, digging indigenous microbial resources is of great significance to cold environment pollution remediation. In this study, we combined single-cell isolation and resuscitation-promoting factor addition. Droplet microfluidic was used to isolate single cells and resuscitation-promoting factor was added during the culture process. In this way, potentially low-temperature biphenyl-degrading strains were isolated from the surface soil samples at different altitudes of the Qinghai-Tibet Plateau. We also investigated the response of isolated strains to resuscitation-promoting factor. A total of 24 strains that could grow at low temperature (15°C) with biphenyl as the sole carbon source were obtained, among which 18 strains were from the resuscitation-promoting factor addition group. Resuscitation-promoting factor addition significantly improved the efficiency of functional bacteria screen. The obtained strains belonged to 9 genera including <italic>Microbacterium</italic>. Most of the obtained strains showed obvious response to resuscitation-promoting factor, and some strains showed marked resuscitation-promoting factor dependence indicating that they almost did not grow without resuscitation-promoting factor addition. This demonstrates the mechanism by which the resuscitation-promoting factor improves single-cell isolation for functional bacteria. The successful isolation of potential low-temperature biphenyl-degrading bacteria from the Qinghai-Tibet Plateau provides the microbial resources for the subsequent prevention and control of biphenyl pollution in the Qinghai-Tibet Plateau and other cold areas. This study presents a new method for isolating and screening of functional bacteria through the combination of resuscitation-promoting factor and single-cell isolation. This method has high efficiency, low cost, and a broad application spectrum. It is a new method for the mining of “microbial dark matter”.

Dimethyl fumarate eliminates differentially culturable Mycobacterium tuberculosis in an intranasal murine model of tuberculosis.

Tuberculosis (TB) claims nearly 1.5 million lives annually. Current TB treatment requires a combination of several drugs administered for at least 6 months. Mycobacterium tuberculosis (Mtb), the causative agent of TB, can persist in infected humans and animals for decades. Moreover, during infection, Mtb produces differentially culturable bacteria (DCB) that do not grow in standard media but can be resuscitated in liquid media supplemented with sterile Mtb culture filtrates or recombinant resuscitation-promoting factors (Rpfs). Here, we demonstrate that, in an intranasal murine model of TB, Mtb DCB are detectable in the lungs after 4 weeks of infection, and their loads remain largely unchanged during a further 8 weeks. Treatment of the infected mice with dimethyl fumarate (DMF), a known drug with immunomodulatory properties, for 8 weeks eliminates Mtb DCB from the lungs and spleens. Standard TB treatment consisting of rifampicin, isoniazid, and pyrazinamide for 8 weeks reduces Mtb loads by nearly four orders of magnitude but does not eradicate DCB. Nevertheless, no DCB can be detected in the lungs and spleens after 8 weeks of treatment with DMF, rifampicin, isoniazid, and pyrazinamide. Our data suggest that addition of approved anti-inflammatory drugs to standard treatment regimens may improve TB treatment and reduce treatment duration.

Promoting Resuscitation of the VBNC Bacteria and Enrichment of Naphthalene-degrading Bacteria from Oil-contaminated Soils with the Rpf4 from Rhodococcus erythropolis KB1

Nowadays, more than 90% of bacteria in nature are in a viable but not culturable (VBNC) state, and they cannot be separated by traditional plate culture methods. However, resuscitation-promoting factors (Rpf) participate in recovery of dormant cells and promoting bacterial growth. The Rpf4 of Rhodococcus erythropolis KB1 is a Rpf like protein of Micrococcus luteus and contained the Rpf domain and LysM domain. In this study, an expression vector for the rpf4 gene was constructed and expressed in Escherichia coli. The recombinant protein Rpf4 was purified by Ni2+-affinity chromatography. The recombinant protein showed a muralytic activity of 549 ± 28.77 U/mg. The purified Rpf4 could increase R. erythropolis KB1 cell proliferation and improve the resuscitation of R. erythropolis cells that were VBNC. The Rpf4 altered the diversity of naphthalene-degrading bacteria enriched from oil-contaminated soils. Based on the high-throughput sequencing, we found that Proteobacteria and Actinobacteria phyla increased from 76.10% and 0.029% to 98.23% and 0.401% after the Rpf4 treatment, while the Bacteroidetes phylum decreased from 22.70% to 0.76%. The genera Enterobacter, Citrobacter, and Acinetobacter increased from 0.0054%, 0.001%, and 0.745% to 19.02%, 6.34% and 2.49%. Some new genera such as Bifidobacterium, Limnobacter, Alloprevotella, Hydrogenophaga, and Nevskia were founded in the treatment group. Meanwhile, 23 naphthalene-degrading bacteria were isolated by adding the Rpf4 protein to the oil-contaminated soil samples. Study on the naphthalene-degrading bacteria will provide a basis for the microbial degradation of naphthalene and other polycyclic aromatic hydrocarbons and discover the wider application value of Rpf.

Use of DosR and Rpf antigens from Mycobacterium tuberculosis to screen for latent and relapse tuberculosis infection in a tuberculosis endemic community of Huainan City.

The dormancy survival regulator (DosR) antigens upgraded during latency and resuscitation-promoting factors (Rpfs) expressed over the reactivation from dormant Mycobacterium tuberculosis (M. tuberculosis) could be used to diagnose tuberculosis (TB) at different stages. We performed a retrospective cohort study based on four groups, including healthy controls (HCs), active tuberculosis infections (ATBs), latent tuberculosis infections (LTBIs), and relapse tuberculosis infections (RTBs) enrolled between November 2020 and June 2021. Compared to the fusion protein E6-C10, combined with early secreted antigenic target 6kDa (ESAT-6) and culture filtrate of 10kDa (CFP-10), the DosR- or Rpf-encoded antigens could not elicit significant IFN-γ concentration for the diagnosis of ATB. Of note, the DosR antigens produce significantly more antigen-specific IFN-γ in LTBIs than Rpfs, and the levels of antigen-specific IFN-γ elicited in RTBs stimulated by Rpfs were higher than the DosR antigens. Among the DosR antigens, Rv2003c was the most immunogenic in diagnosing LTBIs, followed by Rv2007c and Rv2005c. As far as Rpfs are concerned, Rv0867c was the best antigen to identify RTBs, followed by Rv2389c and Rv1009. Both Rv2450c and Rv1884c showed relatively limited IFN-γ concentration in RTBs. Besides, the selected DosR antigens and Rpfs showed ideal specificity and inadequate sensitivity, which could have been enhanced by the fusion antigens prepared by the DosR antigens or Rpfs, respectively. The results of this study can provide more accurate detection methods for LTBIs and RTBs and could be used for screening the dormant M. tuberculosis throughout reactivation.

Rapid Detection of Mycobacterium tuberculosis in Pleural Fluid Using Resuscitation-Promoting Factor-Based Thin Layer Agar Culture Method.

BackgroundPleural tuberculous is difficult to diagnose. Culture is still considered the gold standard, especially in resource-limited settings where quick, cheap, and easy techniques are needed. The aim of the study was to evaluate resuscitation-promoting factors (Rpfs)-based thin layer agar (TLA) culture method for quick detection of Mycobacterium tuberculosis in pleural fluid.MethodsPatients with suspected pleural TB were enrolled prospectively in our hospital, pleural fluid of all patients were collected, stained with Ziehl–Neelsen for acid-fast bacilli (AFB), cultured on Rpfs-TLA, TLA, and Löwenstein–Jensen (LJ) medium, and identified according to recommended procedures.ResultsA total of 137 suspected pleural TB were enrolled and categorized, including 103 pleural TB (49 confirmed and 54 probable pleural TB) and 34 non-TBP patients. The sensitivity of Rpfs-TLA for total pleural TB was 43.7% (34.5∼53.3%), higher than that of TLA 29.1% (21.2∼38.5%) and LJ 26.2% (18.7∼35.5%) (p < 0.01), and all specificity was 100% in the diagnosis of pleural TB. Median time to detection of a positive culture was 11.8 days (95% CI 10.4∼13.4) for Rpfs-TLA, 21.0 days (95% CI 19.1∼22.9) for TLA, and 30.5 days (95% CI 28.5∼32.5) for LJ (p < 0.001).ConclusionRpfs-TLA is an accurate, rapid, cheap, and easy culture method, which makes it promising for use in clinical laboratories.

Promoting effect of the recombinant resuscitation promoting factors-2 of Rhodococcus erythropolis on petroleum degradation and cultivable bacterial diversities of the oil-contaminated soils.

Resuscitation-promoting factors (Rpfs) belong to peptidoglycan hydrolases, which participate in recovery of dormant cells and promoting bacteria growth. In this study, the resuscitation promoting factor rpf2 gene of Rhodococcus erythropolis KB1 was expressed in Escherichia coli and purified by Ni2+ affinity chromatography. The purified recombinant fusion protein Rpf2 showed a closely 50 kDa band on sodium dodecyl sulphate polyacrylamide gel electrophoresis. The protein showed muralytic activity, with a specific activity of 1503 ± 123 U mg-1 when determined with 4-methylumbelliferyl-β-d-N, N',N″-triacetotri-ylchitoside as substrate. It also showed protease activity when measured with azocasein as substrate, with a specific activity of 1528 ± 411 U mg-1 . The addition of the recombinant Rpf2 protein significantly increased petroleum degradation efficiency of the indigenous micro-organisms and the petroleum degradation rates increased from 30·86 to 43·45%, 45·20 and 49·23% in the treatment groups. The recombinant protein also increased the petroleum-degrading bacterial diversities enriched from the contaminated soils. The cultivable bacterial flora of the treatment groups supplemented with different concentrations of Rpf2 increased from 82 genera in 9 phyla to 116 genera in 16 phyla and 138 genera in 16 phyla respectively. Thirteen extra petroleum-degrading bacteria strains were isolated from the petroleum-contaminated soils in the groups containing the recombinant Rpf2.

The Impact of Protease during Recovery from Viable but Non-Culturable (VBNC) State in Vibrio cholerae.

Vibrio cholerae can survive cold stress by entering into a viable but non-culturable (VBNC) state, and resuscitation can be induced either by temperature upshift only or the addition of an anti-dormancy stimulant such as resuscitation-promoting factors (Rpfs) at suitable temperature. In this study, the role of proteinase K was analyzed as an Rpf in V. cholerae. A VBNC state was induced in V. cholerae AN59 in artificial seawater (ASW) media at 4 °C, and recovery could be achieved in filtered VBNC microcosm, called spent ASW media, merely by a temperature upshift to 37 °C. The resuscitation ability of spent ASW was further enhanced by the addition of proteinase K. The mode of action of proteinase K was investigated by comparing its effect on the growth of the VBNC and culturable state of V. cholerae in ASW and spent ASW media. The presence of proteinase K allowed culturable cells to grow faster in ASW by reducing the generation time. However, this effect of proteinase K was more pronounced in stressed VBNC cells. Moreover, proteinase K-supplemented spent ASW could also accelerate the transition of VBNC into recovered cells followed by rapid growth. Additionally, we found that dead bacterial cells were the substrate on which proteinase K acts to support high growth in spent ASW. So, the conclusion is that the proteinase K could efficiently promote the recovery and growth of dormant VBNC cells at higher temperatures by decreasing the duration of the initial lag phase required for transitioning from the VBNC to recovery state and increasing the growth rate of these recovered cells.

Exploitation of extracellular organic matter from Micrococcus luteus to enhance ex situ bioremediation of soils polluted with used lubricants

Chronic pollution by used lubricant oils (ULOs) poses a serious challenge to the environment. Under stress conditions, microorganisms, including potential degraders, can enter a viable but non-culturable (VBNC) state, complicating the bioremediation of ULO-polluted areas. Resuscitation-promoting factors (Rpfs) can reverse this transition and/or enhance the biodegradation performance of both native and augmented strains. Here, Rpf-containing extracellular organic matter (EOM) from Micrococcus luteus was used to enhance the ex situ ULO removal in biostimulated and bioaugmented (with Rhodococcus qingshengii KAG C, R. erythropolis PR4) soils. ULO bioconversion, microbial activity, and CFUs were significantly higher in EOM-treated soils compared to corresponding control soils. After 60 days, the initial ULO concentration (52,500 mg kg−1) was reduced by 37% and 45% with EOM-supplemented biostimulation and bioaugmentation, respectively. Based on high-throughput 16S rRNA analysis, the enhancement was attributable both to the reactivation of EOM-responsive hydrocarbonoclastic bacterial genera (e.g., Pseudomonas, Comamonas, Stenotrophomonas, Gordonia) and to the long-term positive effect of EOM on the degradative efficacy of the introduced rhodococci. Ecotoxicological responses revealed that reduced ULO concentration did not correlate with decreased soil toxicity. Our findings provide an insight into the applicability of EOM in bioremediation and its effects on the soil microbial activity and community composition.

Community-level dormancy potential regulates bacterial beta-diversity succession during the co-composting of manure and crop residues

This study aimed to disclose the bacterial diversity succession during the co-composting of manure and crop residues and to provide new insight into the role of community-level dormancy potential in diversity succession. Illumina sequencing and PICRUSt-estimated metagenomes were used for this purpose. The bacterial richness and phylogenetic diversity decreased in the early and middle stages of composting and were maintained to a stable status in the late stage. Both composting phases and raw materials impacted the aforementioned alpha diversity significantly, while the composting phases had a greater (80%–94%) impact than the raw materials (1%–18%). Bacterial beta-diversity succession exhibited selectivity as the composting proceeded, and the dominant taxa changed into salt- and heat-resistant genera such as Bacillus, Glycomyces, and Halocella. Meanwhile, Georgenia, Actinomadura, and Ruminofilibacter were identified as the dominant predictor taxa of bacterial community succession in composting. Roughly, the abundance of genes underlying dormancy strategies, including sporulation factors (spo0A gene), toxin–antitoxin systems (dinJ/yafP, mazF/E, hipA/O, and relA/E genes), and resuscitation-promoting factors (rpfC gene), increased as composting proceeded and reached the highest in the thermophilic or maturation phases. Co-occurring relationships between bacterial communities and genes underlying dormancy strategies in different composting phases comprised multiple associations dominated by positive edges (50%–97%). The stability in genes underlying dormancy strategies and aggregate dormancy potential had a positive linear correlation with that in bacterial beta diversity (R2 = 0.26–0.42; P < 0.05), but not related significantly to that in richness and phylogenetic diversity. This study highlighted the importance of understanding how community-level dormancy strategies mediated microbial succession in composting to better predict compost maturity and product quality.

A novel strategy for enhancing anaerobic biodegradation of an anthraquinone dye reactive blue 19 with resuscitation-promoting factors

Anaerobic process has been widely applied as a cost-effective method for textile wastewater treatment. However, many bacteria exhibit low metabolic activity in unfavorable conditions due to the entry into a viable but non-culturable (VBNC) state. Thus, in this study, a novel method of using resuscitation-promoting factors (Rpfs), which has been proven to resuscitate and stimulate the growth of VBNC bacteria, is explored to enhance the degradation of the anthraquinone dye reactive blue 19 (RB19) in the anaerobic process. The results show that Rpfs could efficiently prompt RB19 decolorization. Compared to the conventional anaerobic condition, RB19 decolorization efficiency was increased by more than 20% with the Rpf addition. UV–visible spectral and gas chromatograph-mass spectrometry analysis indicate that the aromatic amines structures of RB19 was cleaved. More importantly, the Rpf addition appeared to stimulate and/or enrich some dye-degrading species of the family Peptostreptococcaceae, thus leading to a higher RB19 decolorization efficiency.

Roles of LysM and LytM domains in resuscitation-promoting factor (Rpf) activity and Rpf-mediated peptidoglycan cleavage and dormant spore reactivation

Bacterial dormancy can take many forms, including formation of Bacillus endospores, Streptomyces exospores, and metabolically latent Mycobacterium cells. In the actinobacteria, including the streptomycetes and mycobacteria, the rapid resuscitation from a dormant state requires the activities of a family of cell-wall lytic enzymes called resuscitation-promoting factors (Rpfs). Whether Rpf activity promotes resuscitation by generating peptidoglycan fragments (muropeptides) that function as signaling molecules for spore germination or by simply remodeling the dormant cell wall has been the subject of much debate. Here, to address this question, we used mutagenesis and peptidoglycan binding and cleavage assays to first gain broader insight into the biochemical function of diverse Rpf enzymes. We show that their LysM and LytM domains enhance Rpf enzyme activity; their LytM domain and, in some cases their LysM domain, also promoted peptidoglycan binding. We further demonstrate that the Rpfs function as endo-acting lytic transglycosylases, cleaving within the peptidoglycan backbone. We also found that unlike in other systems, Rpf activity in the streptomycetes is not correlated with peptidoglycan-responsive Ser/Thr kinases for cell signaling, and the germination of rpf mutant strains could not be stimulated by the addition of known germinants. Collectively, these results suggest that in Streptomyces, Rpfs have a structural rather than signaling function during spore germination, and that in the actinobacteria, any signaling function associated with spore resuscitation requires the activity of additional yet to be identified enzymes.

A new multi-epitope peptide vaccine induces immune responses and protection against Leishmania infantum in BALB/c mice.

Visceral leishmaniasis (VL) is a tropical and subtropical disease which is endemic in more than eighty countries aroundthe world. Leishmania infantum is one of the main causative agents of VL disease. Currently, there is no approved-to-market vaccine for VL therapy. In this study, we evaluated cellular and humoral immune responses induced by our newly designed multi-epitope vaccine in BALB/c mice. Four antigenic proteins, including histone H1, sterol 24-c-methyltransferase (SMT), Leishmania-specific hypothetical protein (LiHy), and Leishmania-specific antigenic protein (LSAP) were chosen for the prediction of potential immunodominant epitopes. Moreover, to enhance vaccine immunogenicity, two toll-like receptors 4(TLR4) agonists, resuscitation-promoting factors of Mycobacterium tuberculosis (RpfE and RpfB), were employed as the built-in adjuvants. Immunization with the designed multi-epitope vaccine elicited a robust Th1-type immune response, compared to other groups, as shown by increased levels of IL-2, IFN-γ, TNF-α, and IgG2a. Furthermore, a significant decrease was observed in Th-2-type-related cytokines such as IL-4 in immunized mice. The designed construct also induced a significant reduction in parasite load (p < 0.0001), conferring protection against L. infantum challenge. This study could be promising in gaining insight towards the potential of peptide epitope-based vaccines as effective protective approaches against Leishmania species.

Differential Culturability of Mycobacterium tuberculosis in Culture-Negative Sputum of Patients With Pulmonary Tuberculosis and in a Simulated Model of Dormancy.

Tuberculosis (TB) remains a leading killer among infectious diseases of humans worldwide. Delayed diagnosis is a crucial problem in global TB control programs. Bacteriological methods currently used to diagnose TB in endemic countries take up to 8 weeks, which poses a significant delay in starting antibiotic therapy. The presence of a heterogeneous population of Mycobacterium tuberculosis, the causative agent of TB, is among the reasons for delayed diagnosis by bacteriological methods. Previously, it has been shown that mycobacterial resuscitation-promoting factors (RPFs), a family of proteins secreted by actively growing bacteria into the media, are capable of activating the growth of dormant bacteria, thus enhancing the detection of bacilli in the sputum of confirmed TB cases. However, the variability in bacterial resuscitation by RPF in the sputum of suspected pulmonary TB cases that showed differential smear and/or culture positivity during diagnosis has not been fully explored. Here, we report the presence of non-replicating bacteria in the sputum of suspected TB cases that show differential growth response to RPF treatment. Using crude and recombinant RPF treatment, we show improved sensitivity and reduced time to detect bacilli in the sputum samples of smear-positive/culture-negative or smear-negative/culture-negative cases. We also report the phenotypic heterogeneity in the RPF responsiveness among Mtb strains using an in vitro dormancy model. Our findings have implications for improving the bacteriological diagnostic modalities currently used to diagnose TB in endemic countries.

Enzyme characterization and biological activities of a resuscitation promoting factor from an oil degrading bacterium Rhodococcus erythropolis KB1.

Resuscitation-promoting factors (Rpf) are a class of muralytic enzymes, which participate in recovery of dormant cells and promoting bacteria growth in poor media. In the present study the expression vector of the rpf-1 gene from an oil-degrading bacterium Rhodococcus erythropolis KB1 was constructed and expressed in Escherichia coli. The expressed protein was purified by Ni2+-affinity chromatography, and showed muralytic activity when measured with 4-methylumbelliferyl-β-D-N,N′,N″-triacetyl chitotrioside as substrate. Addition of purified Rpf-1 to R. erythropolis culture efficiently improved bacterial cell growth. The purified protein also increased resuscitation of viable but nonculturable cells of R. erythropolis to culturable state. The conserved amino acid residues including Asp45, Glu51, Cys50, Thr60, Gln69, Thr74, Trp75 and Cys114 of the Rpf-1 were replaced with different amino acids. The mutant proteins were also expressed and purified with Ni2+-affinity chromatography. The muralytic activities of the mutant proteins decreased to different extents when compared with that of the wild type Rpf-1. Gln69 was found to play the most important role in the enzyme activity, substitution of Gln69 with lysine (Q69K) resulted in the greatest decrease of muralytic activity. The other amino acid residues such as Asp45, Glu51, Cys50 and Cys114 were also found to be very important in maintaining muralytic activity and biological function of the Rpf-1. Our results indicated that Rpf-1 from R. erythropolis showed muralytic activities and weak protease activity, but the muralytic activity was responsible for its growth promotion and resuscitation activity.