Unravelling bacterial virulence factors in yeast: From identification to the elucidation of their mechanisms of action.

Pathogenic bacteria employ virulence factors (VF) to establish infection and cause disease in their host. Yeasts, Saccharomyces cerevisiae and Saccharomyces pombe, are useful model organisms to study the functions of bacterial VFs and their interaction with targeted cellular processes because yeast processes and organelle structures are highly conserved and similar to higher eukaryotes. In this review, we describe the principles and applications of the yeast model for the identification and functional characterisation of bacterial VFs to investigate bacterial pathogenesis. The growth inhibition phenotype caused by the heterologous expression of bacterial VFs in yeast is commonly used to identify candidate VFs. Then, subcellular localisation patterns of bacterial VFs can provide further clues about their target molecules and functions during infection. Yeast knockout and overexpression libraries are also used to investigate VF interactions with conserved eukaryotic cell structures (e.g., cytoskeleton and plasma membrane), and cellular processes (e.g., vesicle trafficking, signalling pathways, and programmed cell death). In addition, the yeast growth inhibition phenotype is also useful for screening new drug leads that target and inhibit bacterial VFs. This review provides an updated overview of new tools, principles and applications to study bacterial VFs in yeast.

Exploring the role of impedance spectroscopy in assessing 405 nm laser-induced inactivation of saccharomyces cerevisiae.

Impedance spectroscopy was employed to assess the electrical properties of yeast following 405 nm laser irradiation, exploring the effects of visible, non-ionizing laser-induced inactivation as a more selective and safer alternative for photoinactivation applications compared to the use of DNA targeting, ionizing UV light. Capacitance and impedance spectra were obtained for yeast suspensions irradiated for 10, 20, 30, and 40 min using 100 and 200 mW laser powers. Noticeable differences in capacitance spectra were observed at lower frequencies (40 Hz to 1 kHz), with a significant increase at 40 min for both laser powers. ß-dispersion was evident in the impedance spectra in the frequency range of 10 kHz to 10 MHz. The characteristic frequency of dielectric relaxation steadily shifted to higher frequencies with increasing irradiation time, with a drastic change observed at 40 min for both laser powers. These changes signify a distinct alteration in the physical state of yeast. A yeast spot assay demonstrated a decrease in cell viability with increasing laser irradiation dose. The results indicate a correlation between changes in electrical properties, cell viability, and the efficacy of 405 nm laser-induced inactivation. Impedance spectroscopy is shown to be an efficient, non-destructive, label-free method for monitoring changes in cell viability in photobiological effect studies. The development of impedance spectroscopy-based real-time studies in photoinactivation holds promise for advancing our understanding of light-cell interactions in medical applications.

Identifying Leptospira interrogans putative virulence factors with a yeast protein expression screen.

Leptospirosis is a zoonotic disease caused by pathogenic Leptospira spp., with global implications primarily in tropical countries. However, the mechanisms of leptospiral pathogenesis are still not fully known and not all virulence factors (VFs) have been identified. Budding yeast, Saccharomyces cerevisiae is a popular eukaryotic model which has been used to identify bacterial VFs that target the conserved eukaryotic cellular processes. In this study, we screened for putative VFs of L. interrogans, one of the dominant species causing leptospirosis, by expressing candidate VFs in budding yeast and examining their impact on yeast growth in a high-throughput format. From an initial selection of 288 L. interrogans ORFs, we screened 226 candidate VFs in a yeast growth inhibition assay and identified nine putative VFs in four categories (adhesion, enzymatic, host structure interaction, and immunogenicity). Notably, LIC10280 was highly toxic even when expressed at low copies. We also observed specific subcellular localization for several putative VFs. This study shows that there are still potential L. interrogans VFs that await discovery. KEY POINTS: • High-throughput cloning and expression of leptospiral proteins in yeast. • Heterologous expression of nine leptospiral proteins inhibited yeast growth. • An uncharacterized protein LIC10280 maybe a putative VF for further validation.

Evaluation of Salmonella Typhi antigen YncE alongside HlyE for the detection of typhoid fever and its carriers.

Typhoid fever is a disease caused by Salmonella Typhi that was implicated in millions of illnesses worldwide annually. Individuals that do not recover fully from typhoid fever can become asymptomatic carriers of the disease. Host antibodies against the S. Typhi antigens, HlyE (for acute typhoid) and YncE (for carriers) were previously reported to be useful biomarkers for the disease. Here, we expressed and purified recombinant HlyE and YncE antigens and tested the IgG, IgA and IgM responses in 422 sera samples retrieved from acute typhoid patients, other febrile, food handlers, and healthy individuals. The results showed that HlyE-IgG, -IgA and -IgM ELISAs have a collective sensitivity of 83% while YncE-IgG and -IgA ELISAs identified 16 possible carriers based on their antibody profiles. The identification of sensitive biomarkers for typhoid carrier detection is crucial for disease eradication.

Multi-isotype antibody responses against the multimeric Salmonella Typhi recombinant hemolysin E antigen.

The detection and measurement of different antibody isotypes in the serum provide valuable indicators of the different stages of typhoid infection. Here, the ability of S. Typhi recombinant hemolysin E (HlyE) to detect multi-isotype antibody responses in sera of patients with typhoid and paratyphoid A was investigated using an indirect antibody immunoassay. Nanogram amounts of HlyE were found to be sufficient for detection of IgG and IgA isotypes and, in a study of individuals' sera (n = 100), the immunoassay was able to distinguish between typhoid and non-typhoid sera. The overall sensitivity, specificity and efficiency of the ELISA were 70% (39/56), 100% (44/44) and 83% respectively.

Principles and application of antibody libraries for infectious diseases.

Antibodies have been used efficiently for the treatment and diagnosis of many diseases. Recombinant antibody technology allows the generation of fully human antibodies. Phage display is the gold standard for the production of human antibodies in vitro. To generate monoclonal antibodies by phage display, the generation of antibody libraries is crucial. Antibody libraries are classified according to the source where the antibody gene sequences were obtained. The most useful library for infectious diseases is the immunized library. Immunized libraries would allow better and selective enrichment of antibodies against disease antigens. The antibodies generated from these libraries can be translated for both diagnostic and therapeutic applications. This review focuses on the generation of immunized antibody libraries and the potential applications of the antibodies derived from these libraries.

TolC facilitates the intracellular survival and immunomodulation of Salmonella Typhi in human host cells.

Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever, a systemic infection that affects millions of people worldwide. S. Typhi can invade and survive within host cells, such as intestinal epithelial cells and macrophages, by modulating their immune responses. However, the immunomodulatory capability of S. Typhi in relation to TolC-facilitated efflux pump function remains unclear. The role of TolC, an outer membrane protein that facilitates efflux pump function, in the invasion and immunomodulation of S. Typhi, was studied in human intestinal epithelial cells and macrophages. The tolC deletion mutant of S. Typhi was compared with the wild-type and its complemented strain in terms of their ability to invade epithelial cells, survive and induce cytotoxicity in macrophages, and elicit proinflammatory cytokine production in macrophages. The tolC mutant, which has a defective outer membrane, was impaired in invading epithelial cells compared to the wild-type strain, but the intracellular presence of the tolC mutant exhibited greater cytotoxicity and induced higher levels of proinflammatory cytokines (IL-1ß and IL-8) in macrophages compared to the wild-type strain. These effects were reversed by complementing the tolC mutant with a functional tolC gene. Our results suggest that TolC plays a role in S. Typhi to efficiently invade epithelial cells and suppress host immune responses during infection. TolC may be a potential target for the development of novel therapeutics against typhoid fever.

Phage display antibodies for diagnostic applications.

With major developments in molecular biology, numerous display technologies have been successfully introduced for recombinant antibody production. Even so, phage display still remains the gold standard for recombinant antibody production. Its success is mainly attributed to the robust nature of phage particles allowing for automation and adaptation to modifications. The generation of monospecific binders provides a vital tool for diagnostics at a lower cost and higher efficiency. The flexibility to modify recombinant antibodies allows great applicability to various platforms for use. This review presents phage display technology, application and modifications of recombinant antibodies for diagnostics.

Deletion of Salmonella enterica serovar Typhi tolC reduces bacterial adhesion and invasion toward host cells.

Background: S. Typhi is a Gram-negative bacterium that causes typhoid fever in humans. Its virulence depends on the TolC outer membrane pump, which expels toxic compounds and antibiotics. However, the role of TolC in the host cell adhesion and invasion by S. Typhi is unclear. Objective: We aimed to investigate how deleting the tolC affects the adhesion and invasion of HT-29 epithelial and THP-1 macrophage cells by S. Typhi in vitro. Methods: We compared the adhesion and invasion rates of the wild-type and the tolC mutant strains of S. Typhi using in vitro adhesion and invasion assays. We also measured the expression levels of SPI-1 genes (invF, sipA, sipC, and sipD) using quantitative PCR. Results: We found that the tolC mutant showed a significant reduction in adhesion and invasion compared to the wild-type strain in both cell types. We also observed that the expression of SPI-1 genes was downregulated in the tolC mutant. Discussion: Our results suggest that TolC modulates the expression of SPI-1 genes and facilitates the adhesion and invasion of host cells by S. Typhi. Our study provides new insights into the molecular mechanisms of S. Typhi pathogenesis and antibiotic resistance. However, our study is limited by the use of in vitro models and does not reflect the complex interactions between S. Typhi and host cells in vivo.

Intensification of Inclusion Body Processing via Temperature-Based Refolding.

Inclusion bodies (IB) are dense insoluble aggregates of mostly misfolded polypeptides that usually result from recombinant protein overexpression. IB formation has been observed in protein expression systems such as E. coli, yeast, and higher eukaryotes. To recover soluble recombinant proteins in their native state, IB are commonly first solubilized with a high concentration of denaturant. This is followed by concurrent denaturant removal or reduction and a transition into a refolding-favorable chemical environment to facilitate the refolding of solubilized protein to its native state. Due to the high concentration of denaturant used, conventional refolding approaches can result in dilute products and are buffer inefficient. To circumvent the limitations of conventional refolding approaches, a temperature-based refolding approach which combines a low concentration of denaturant (0.5 M guanidine hydrochloride, GdnHCl) with a high temperature (95 °C) during solubilization was proposed. In this chapter, we describe a temperature-based refolding approach for the recovery of core streptavidin (cSAV) from IB. Through the temperature-based approach, intensification was achieved through the elimination of a concentration step which would be required by a dilution approach and through a reduction in buffer volumes required for dilution or denaturant removal. High-temperature treatment during solubilization may have also resulted in the denaturation and aggregation of undesired host-cell proteins, which could then be removed through a centrifugation step resulting in refolded cSAV of high purity without the need for column purification. Refolded cSAV was characterized by biotin-binding assay and SDS-PAGE, while purity was determined by RP-HPLC.

Vipirinin, a coumarin-based HIV-1 Vpr inhibitor, interacts with a hydrophobic region of VPR.

The human immunodeficiency virus 1 (HIV-1) viral protein R (Vpr) is an accessory protein that has been shown to have multiple roles in HIV-1 pathogenesis. By screening chemical libraries in the RIKEN Natural Products Depository, we identified a 3-phenyl coumarin-based compound that inhibited the cell cycle arrest activity of Vpr in yeast and Vpr-dependent viral infection of human macrophages. We determined its minimal pharmacophore through a structure-activity relationship study and produced more potent derivatives. We detected direct binding, and by assaying a panel of Vpr mutants, we found the hydrophobic region about residues Glu-25 and Gln-65 to be potentially involved in the binding of the inhibitor. Our findings exposed a targeting site on Vpr and delineated a convenient approach to explore other targeting sites on the protein using small molecule inhibitors as bioprobes.

RelEB3 toxin-antitoxin system of Salmonella Typhimurium with a ribosome-independent toxin and a mutated non-neutralising antitoxin.

The RelEB3 toxin-antitoxin (TA) system of Salmonella enterica subsp. enterica serovar Typhimurium consists of a RelE3 toxin which suppresses bacterial growth, but its RelB3 antitoxin does not neutralise the toxin. The relEB3 operon is widespread in Proteobacteria and is related to higBA2 from Vibrio cholerae. In contrast to the ribosome-dependent HigB2 toxin, however, the RelE3 toxin degraded free RNA independently of the ribosome. A basic loop possibly involved in HigB2's binding to the ribosome is shortened in RelE3, which instead contains a uniquely conserved R51 important for RelE3's toxicity. The RelB3 antitoxin, meanwhile, specifically recognised the CACCTGGTG palindromic motif in the promoter site. RelB3 contains a unique P14 which is conserved as Ala in most homologues, and mutating P14 to Ala enabled the antitoxin to bind to RelE3 and restored bacterial growth. The P14 RelB3 variant, which most likely arose by a point mutation in a recent ancestor of S. Typhimurium and closely related serovars, could have possibly provided the bacteria with a faster response to stress, and might have spread to other serovars through homologous recombination.

Enhancing yeast growth with carboxylates under multiple nutrient limitations.

Yeast cell death is triggered when essential nutrients such as potassium and lipid are limited but ammonium is in excess. When ammonium and glucose were maintained at 100% of the normal concentration while all the other essential nutrients in yeast nitrogen base (YNB) were reduced to 2%, yeast growth was halted by ammonium toxicity. Yeast started to grow again when either ammonium was also reduced to 2% or gluconate was added, but simultaneously adding gluconate as well as reducing all the nutrients except glucose 50-fold revived yeast growth to a greater extent, i.e. a quarter of the normal growth. Gluconate, as well as formate and alginate, stimulated yeast growth by buffering the drop in pH. Yeast cells were seemingly more susceptible to low pH under the nutrient-limited conditions, entering the stationary phase at pH higher than that of the normal condition. Carboxylate salts may prove a cost-efficient replacement for large proportions of the essential nutrients as yeast cells, in the presence of 2 mg ml-1 gluconate, could still achieve nearly 90% of the normal growth when cultured in only 10% of the normal YNB concentration.

Screening of selected ageing-related proteins that extend chronological life span in yeast Saccharomyces cerevisiae.

Ageing-related proteins play various roles such as regulating cellular ageing, countering oxidative stress, and modulating signal transduction pathways amongst many others. Hundreds of ageing-related proteins have been identified, however the functions of most of these ageing-related proteins are not known. Here, we report the identification of proteins that extended yeast chronological life span (CLS) from a screen of ageing-related proteins. Three of the CLS-extending proteins, Ptc4, Zwf1, and Sme1, contributed to an overall higher survival percentage and shorter doubling time of yeast growth compared to the control. The CLS-extending proteins contributed to thermal and oxidative stress responses differently, suggesting different mechanisms of actions. The overexpression of Ptc4 or Zwf1 also promoted rapid cell proliferation during yeast growth, suggesting their involvement in cell division or growth pathways.

Identification of Tropical Plant Extracts That Extend Yeast Chronological Life Span.

Certain plant extracts (PEs) contain bioactive compounds that have antioxidant and lifespan-extending activities on organisms. These PEs play different roles in cellular processes, such as enhancing stress resistance and modulating longevity-defined signaling pathways that contribute to longevity. Here, we report the discovery of PEs that extended chronological life span (CLS) in budding yeast from a screen of 222 PEs. We identified two PEs, the leaf extracts of Manihot esculenta and Wodyetia bifurcata that extended CLS in a dose-dependent manner. The CLS-extending PEs also conferred oxidative stress tolerance, suggesting that these PEs might extend yeast CLS through the upregulation of stress response pathways.

Genetic relatedness in extended-spectrum beta-lactamase-producing Escherichia coli from clinical isolates using enterobacterial repetitive intergenic consensus polymerase chain reaction.

OBJECTIVES: The aim of this study is to determine the genetic relatedness of extended-spectrum beta-lactamases (ESBL)-producing Escherichia coli using the enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) technique. METHODS: Suspected Gram-negative bacteria with their identities from the clinical samples were confirmed using Microgen GN-A-ID Kit. The double-disc synergy test was used to confirm for ESBL-producing E. coli. The susceptibility of the organisms was tested against eleven antimicrobial agents. A singleplex PCR assay was carried out targeting TEM, SHV, CTX-M, and OXA. ERIC-PCR performed, and band patterns obtained were visually evaluated. A dendrogram of the ERIC-PCR fingerprint pattern was done with the aid of DendroUPGMA using the cluster method. RESULTS: Of the 576 clinical samples collected, 23 isolates were confirmed E. coli, and all (100%) are ESBL producers. The highest antibiotic resistance rate was recorded in cefixime (95.6%), and the least was amikacin (17.4%). The predominant ESBL gene is blaTEM genes (95.6%). Gel analysis of ERIC-PCR revealed 1-6 bands. The profiles of the ERIC-PCR differentiated the 23 E. coli isolates into four ERIC cluster types. CONCLUSION: More than 80% of the isolates are sensitive to amikacin, with greater than 95% harboring blaTEM genes. Overall, ERIC obtained from the clinical specimens indicated some evidence in the genetic relatedness of the ESBL genes among E. coli isolates.

Performance of Immunodiagnostic Tests for Typhoid Fever: A Systematic Review and Meta-Analysis.

Typhoid fever, also known as typhoid, is a life-threatening bacterial infection that remains a global health concern. The infection is associated with a significant morbidity and mortality rate, resulting in an urgent need for specific and rapid detection tests to aid prevention and management of the disease. The present review aims to assess the specificity and sensitivity of the available literature on the immunodiagnostics of typhoid fever. A literature search was conducted using three databases (PubMed, ProQuest and Scopus) and manual searches through the references of identified full texts to retrieve relevant literature published between 1 January 2011 and 31 December 2020. Of the 577 studies identified in our search, 12 were included in further analysis. Lipopolysaccharides (LPS) and hemolysin E (HlyE) were the most frequently studied antigens. The specimens examined in these studies included serum and saliva. Using blood culture as the gold standard, anti-LPS IgA gave the highest sensitivity of 96% (95% CI: 93-99) and specificity of 96% (95% CI: 93-99) for distinguishing between typhoid cases and healthy controls, whereas the combination of anti-LPS and anti-flagellin total IgGAM gave the highest sensitivity of 93% (95% CI: 86-99) and specificity of 95% (95% CI: 89-100) for distinguishing typhoid cases and other febrile infections. A comparably high sensitivity of 92% (95% CI: 86-98) and specificity of 89% (95% CI: 78-100) were shown in testing based on detection of the combination of anti-LPS (IgA and IgM) and anti-HlyE IgG as well as a slightly lower sensitivity of 91% (95% CI: 74-100) in the case of anti-50kDa IgA. Anti-50kDa IgM had the lowest sensitivity of 36% (95% CI: 6-65) against both healthy and febrile controls. The development of a rapid diagnostic test targeting antibodies against lipopolysaccharides combined with flagellin appeared to be a suitable approach for the rapid detection test of typhoid fever. Saliva is added benefit for rapid typhoid diagnosis since it is less invasive. As a result, further studies could be done to develop additional approaches for adopting such samples.

Genomic Instability and Cellular Senescence: Lessons From the Budding Yeast.

Aging is a complex biological process that occurs in all living organisms. Aging is initiated by the gradual accumulation of biomolecular damage in cells leading to the loss of cellular function and ultimately death. Cellular senescence is one such pathway that leads to aging. The accumulation of nucleic acid damage and genetic alterations that activate permanent cell-cycle arrest triggers the process of senescence. Cellular senescence can result from telomere erosion and ribosomal DNA instability. In this review, we summarize the molecular mechanisms of telomere length homeostasis and ribosomal DNA stability, and describe how these mechanisms are linked to cellular senescence and longevity through lessons learned from budding yeast.

Comparison of microplate- and bottle-based methods to age yeast for chronological life span assays.

This study compared the chronological life span and survival of Saccharomyces cerevisiae aged in a microplate or bottle, under different aeration and calorie restriction conditions. Our data shows that limited aeration in the microplate-aged culture contributed to slower outgrowth but extended yeast CLS compared to the bottle-aged culture.