Pan-genomics, drug candidate mining and ADMET profiling of natural product inhibitors screened against Yersinia pseudotuberculosis.

Yersinia pseudotuberculosis belongs to the family Enterobacteriaceae and is responsible for scarlatinoid fever, food poisoning, post-infectious complications like erythema nodosum/reactive arthritis as well as pseudoappendicitis in children. Genome sequences of the 23 whole genomes from NCBI were utilized for conducting the pan-genomic analysis. Essential proteins from the core region were obtained and drug targets were identified using a hierarchal in silico approach. Among these, multidrug resistance protein sub-unit mdtC was chosen for further analysis. This protein unit confers resistance to antibiotics upon forming a tripartite complex with units A and B in Escherichia coli. Details of the function have not yet been elucidated experimentally in Yersinia spp. Computational structure modeling and validation were followed by screening against phytochemical libraries of traditional Indian (Ayurveda), North African, and traditional Chinese flora using Molecular Operating Environment software version 2019.0102. ADMET profiling and descriptor study of best docked compounds was studied. Since phytotherapy is the best resort to antibiotic resistance so these compounds should be tested experimentally to further validate the results. The obtained information could aid wet-lab scientists to work on the scaffold of screened drug-like compounds from natural resources. This could be useful in our quest for antibiotic-resistant therapy against Y. pseudotuberculosis.

Subpopulations of Stressed Yersinia pseudotuberculosis Preferentially Survive Doxycycline Treatment within Host Tissues.

Severe systemic bacterial infections result in colonization of deep tissues, which can be very difficult to eliminate with antibiotics. It remains unclear if this is because antibiotics are not reaching inhibitory concentrations within tissues, if subsets of bacteria are less susceptible to antibiotics, or if both contribute to limited treatment efficacy. To detect exposure to doxycycline (Dox) present in deep tissues following treatment, we generated a fluorescent transcriptional reporter derived from the tet operon to specifically detect intracellular tetracycline exposure at the single bacterial cell level. Dox exposure was detected in the spleen 2 h after intraperitoneal injection, and by 4 h postinjection, this treatment resulted in a significant decrease in viable Yersinia pseudotuberculosis bacteria in the spleen. Nitric oxide-stressed bacteria preferentially survived treatment, suggesting that stress was sufficient to alter Dox susceptibility. Many bacteria (∼10%) survived a single dose of Dox, and the antibiotic accumulated at the periphery of microcolonies to growth inhibitory concentrations until 48 h posttreatment. After this time point, antibiotic concentrations decreased and bacterial growth resumed. Dox-treated mice eventually succumbed to the infection, albeit with significantly prolonged survival relative to that of untreated mice. These results indicate that Dox delivery by intraperitoneal injection results in rapid diffusion of inhibitory concentrations of antibiotic into the spleen, but stressed cells preferentially survive drug treatment, and bacterial growth resumes once drug concentrations decrease. This fluorescent reporter strategy for antibiotic detection could easily be modified to detect the concentration of additional antimicrobial compounds within host tissues following drug administration.IMPORTANCE Bacterial infections are very difficult to treat when bacteria spread into the bloodstream and begin to replicate within deep tissues, such as the spleen. Subsets of bacteria can survive antibiotic treatment, but it remains unclear if this survival is because of limited drug diffusion into tissues, or if there are changes within the bacteria, promoting survival of some bacterial cells. Here, we have developed a fluorescent reporter to detect doxycycline (Dox) diffusion into host tissues, and we show that Dox impacts the bacterial population within hours of administration and inhibits bacterial growth for 48 h. However, bacterial growth resumes when antibiotic concentrations decrease. Subsets of bacteria, stressed by the host response to infection, survive Dox treatment at a higher rate. These results provide critical information about the dynamics that occur within deep tissues following antibiotic administration and suggest that subsets of bacteria are predisposed to survive inhibitory concentrations of antibiotic before exposure.

Exploring resveratrol dimers as virulence blocking agents - Attenuation of type III secretion in Yersinia pseudotuberculosis and Pseudomonas aeruginosa.

Bacterial infections continue to threaten humankind and the rapid spread of antibiotic resistant bacteria is alarming. Current antibiotics target essential bacterial processes and thereby apply a strong selective pressure on pathogenic and non-pathogenic bacteria alike. One alternative strategy is to block bacterial virulence systems that are essential for the ability to cause disease but not for general bacterial viability. We have previously show that the plant natural product (-)-hopeaphenol blocks the type III secretion system (T3SS) in the Gram-negative pathogens Yersinia pseudotuberculosis and Pseudomonas aeruginosa. (-)-Hopeaphenol is a resveratrol tetramer and in the present study we explore various resveratrol dimers, including partial structures of (-)-hopeaphenol, as T3SS inhibitors. To allow rapid and efficient assessment of T3SS inhibition in P. aeruginosa, we developed a new screening method by using a green fluorescent protein reporter under the control of the ExoS promoter. Using a panel of assays we showed that compounds with a benzofuran core structure i.e. viniferifuran, dehydroampelopsin B, anigopreissin A, dehydro-δ-viniferin and resveratrol-piceatannol hybrid displayed significant to moderate activities towards the T3SS in Y. pseudotuberculosis and P. aeruginosa.

Influence of pH on the activity of finafloxacin against extracellular and intracellular Burkholderia thailandensis, Yersinia pseudotuberculosis and Francisella philomiragia and on its cellular pharmacokinetics in THP-1 monocytes.

OBJECTIVES: Burkholderia pseudomallei, Yersinia pestis and Francisella tularensis are facultative intracellular bacteria causing life-threatening infections. We have (a) compared the activity of finafloxacin (a fluoroquinolone in development showing improved activity at acidic pH) with that of ciprofloxacin, levofloxacin and imipenem against the extracellular and intracellular (THP-1 monocytes) forms of infection by attenuated surrogates of these species (B. thailandensis, Y. pseudotuberculosis, F. philomiragia) and (b) assessed finafloxacin cellular pharmacokinetics (accumulation, distribution, efflux). METHODS: Bacteria in broth or in infected monocytes were exposed to antibiotics at pH 7.4 or 5.5 for 24 hr. Maximal relative efficacies (Emax) and static concentrations (Cs) were calculated using the Hill equation (concentration-response curves). Finafloxacin pharmacokinetics in cells at pH 7.4 or 5.5 was investigated using 14C-labelled drug. RESULTS: Extracellularly, all drugs sterilized the cultures, with finafloxacin being two to six times more potent at acidic pH. Intracellularly, Emax reached the limit of detection (4-5 log10 cfu decrease) for finafloxacin against all species, but only against B. thailandensis and F. philomiragia for ciprofloxacin and levofloxacin, while imipenem caused less than 2 log10 cfu decrease for all species. At acid pH, Cs shifted to two to five times lower values for finafloxacin and to one to four times higher values for the other drugs. Finafloxacin accumulated in THP-1 cells by approximately fivefold at pH 7.4 but up to 20-fold at pH 5.5, and distributed in the cytosol. CONCLUSIONS: Fluoroquinolones have proven to be effective in reducing the intracellular reservoirs of B. thailandensis, Y. pseudotuberculosis and F. philomiragia, with finafloxacin demonstrating an additional advantage in acidic environments.

A Recombinant Attenuated Yersinia pseudotuberculosis Vaccine Delivering a Y. pestis YopENt138-LcrV Fusion Elicits Broad Protection against Plague and Yersiniosis in Mice.

In this study, a novel recombinant attenuated Yersinia pseudotuberculosis PB1+ strain (χ10069) engineered with ΔyopK ΔyopJ Δasd triple mutations was used to deliver a Y. pestis fusion protein, YopE amino acid 1 to 138-LcrV (YopENt138-LcrV), to Swiss Webster mice as a protective antigen against infections by yersiniae. χ10069 bacteria harboring the pYA5199 plasmid constitutively synthesized the YopENt138-LcrV fusion protein and secreted it via the type 3 secretion system (T3SS) at 37°C under calcium-deprived conditions. The attenuated strain χ10069(pYA5199) was manifested by the establishment of controlled infection in different tissues without developing conspicuous signs of disease in histopathological analysis of microtome sections. A single-dose oral immunization of χ10069(pYA5199) induced strong serum antibody titers (log10 mean value, 4.2), secretory IgA in bronchoalveolar lavage (BAL) fluid from immunized mice, and Yersinia-specific CD4+ and CD8+ T cells producing high levels of tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), and interleukin 2 (IL-2), as well as IL-17, in both lungs and spleens of immunized mice, conferring comprehensive Th1- and Th2-mediated immune responses and protection against bubonic and pneumonic plague challenges, with 80% and 90% survival, respectively. Mice immunized with χ10069(pYA5199) also exhibited complete protection against lethal oral infections by Yersinia enterocolitica WA and Y. pseudotuberculosis PB1+. These findings indicated that χ10069(pYA5199) as an oral vaccine induces protective immunity to prevent bubonic and pneumonic plague, as well as yersiniosis, in mice and would be a promising oral vaccine candidate for protection against plague and yersiniosis for human and veterinary applications.

Automated multi-sample acquisition and analysis using atomic force microscopy for biomedical applications.

In the last 20 years, atomic force microscopy (AFM) has emerged as a ubiquitous technique in biological research, allowing the analysis of biological samples under near-physiological conditions from single molecules to living cells. Despite its growing use, the low process throughput remains a major drawback. Here, we propose a solution validated on a device allowing a fully automated, multi-sample analysis. Our approach is mainly designed to study samples in fluid and biological cells. As a proof of concept, we demonstrate its feasibility applied to detect and scan both fixed and living bacteria before completion of data processing. The effect of two distinct treatments (i.e. gentamicin and heating) is then evidenced on physical parameters of fixed Yersinia pseudotuberculosis bacteria. The multi-sample analysis presented allows an increase in the number of scanned samples while limiting the user's input. Importantly, cantilever cleaning and control steps are performed regularly-as part of the automated process-to ensure consistent scanning quality. We discuss how such an approach is paving the way to AFM developments in medical and clinical fields, in which statistical significance of results is a prerequisite.

Assessment of the efficacy of an autogenous vaccine against Yersinia pseudotuberculosis in young Merino sheep.

AIMS To assess the efficacy of an autogenous vaccine against Yersinia pseudotuberculosis III in preventing clinical disease and deaths due to yersiniosis in young Merino sheep, and to determine the effect of vaccination on the prevalence of faecal shedding of pathogenic Yersinia spp., daily liveweight gain, and development of antibodies to Yersinia spp. following vaccination and natural exposure. METHODS In six groups (three groups each from two farms) of young Merino sheep, 148-150 animals were systematically allocated to be vaccinated twice with an autogenous, formalin- killed bacterin vaccine containing Y. pseudotuberculosis serotype III or to remain non-vaccinated. All vaccinated and non-vaccinated sheep were run together in their original groups throughout the trial. Faecal and blood samples were collected, and liveweight measured, at the time of vaccination and subsequently over a 6-month period to determine faecal shedding of Y. enterocolitica and Y. pseudotuberculosis, seroprevalence of antibodies to Yersinia outer membrane proteins (YOP) and changes in liveweight. RESULTS None of the six trial groups experienced an outbreak of clinical yersiniosis during the study period. On Farm A, the prevalence of shedding of either or both Yersinia spp. was <40% on all but one sampling occasions. On Farm B the prevalence of shedding of both Yersinia spp. peaked at 98%, 96 days after vaccination. Mean liveweight and daily liveweight gain at the end of the study were similar in vaccinated and non-vaccinated groups on both farms (p>0.1), as was the prevalence of faecal shedding of Yersinia spp. (p>0.2), and the proportion of animals that became seropositive for antibodies to YOP following vaccination (p>0.1). CONCLUSIONS AND CLINICAL RELEVANCE This vaccine had, at most, limited effects on seroconversion and, under the conditions of this study, had no demonstrable impact on liveweight, mean daily liveweight gain or faecal shedding of Yersinia spp. Further studies are needed to determine the efficacy of this vaccine during outbreaks of yersiniosis or following experimental challenge with pathogenic Yersinia spp..

Relationship between Adaptive Changing of Lysophosphatidylethanolamine Content in the Bacterial Envelope and Ampicillin Sensitivity of Yersinia pseudotuberculosis.

The low permeability of porin channels is the possible reason for Gram-negative bacterial resistance to antibiotics. The adaptive accumulation of lysophosphatidylethanolamine (LPE) in Yersinia pseudotuberculosis induces conformational changes of OmpF porin that may hinder the transport of antibiotics through this channel. The present study was aimed to test whether the changes in LPE content affect the resistance of bacteria to ampicillin. The addition of glucose to the culture medium was shown to simultaneously increase the level of LPE and minimum inhibitory concentration (MIC) for ampicillin of Y. pseudotuberculosis cells 6- and 2-fold, respectively. However, the coadministration of glucose and polyphenol extract from buckwheat husks reduced the content of LPE 2-fold and restored MIC to the control value. Thus, PBEH can be used as antibiotic adjuvant to improve an antibiotic's ability to cross the outer membrane. The present work demonstrated: (i) the role of adaptive changes in the lipid composition of Y. pseudotuberculosis in the development of antibiotic resistance, and (ii) the promising use of PBEH in combination therapy to increase the susceptibility of Gram-negative bacteria to the conventional ß-lactam antibiotics, probably attenuating in vivo a previously demonstrated effect of LPE on the conformation and function of the OmpF channel.

Investigation of the Physical and Bioactive Properties of Bromo- and Iodo-Containing Sponge-Derived Compounds Possessing an Oxyphenylethanamine Core.

This research set out to identify compounds from marine sponges that can act as bacterial virulence blockers. Extracts from a total of 80 sponges collected from throughout Indonesia were screened in a high-throughput NF-κB-based screen that identifies compounds capable of inhibiting the bacterial type III secretion system (T3SS) in Yersinia pseudotuberculosis. An extract that was shown to inhibit T3SS-driven NF-κB expression was obtained from an Iotrochota cf. iota sponge and was the source of seven new bromo- and iodo-containing compounds, all of which contain a 2-(4-oxyphenyl)ethan-1-amine core. Five were determined to be new compounds and named enisorines A-E (1-5). The remaining two were determined to be new hemibastadinol analogues named (+)-1-O-methylhemibastadinol 2 (6) and (+)-1-O-methylhemibastadinol 4 (7). All seven compounds inhibited T3SS-dependent YopE secretion and did not affect the growth or metabolic activity of Y. pseudotuberculosis. The most potent inhibitors of T3SS activity were enisorine C (3), enisorine E (5), and (+)-1-O-methylhemibastadinol 2 (6), all of which inhibited YopE secretion by >50% at 30 µM.

Differential impact of lipopolysaccharide defects caused by loss of RfaH in Yersinia pseudotuberculosis and Yersinia pestis.

RfaH enhances transcription of a select group of operons controlling bacterial surface features such as lipopolysaccharide (LPS). Previous studies have suggested that rfaH may be required for Yersinia pseudotuberculosis resistance to antimicrobial chemokines and survival during mouse infections. In order to further investigate the role of RfaH in LPS synthesis, resistance to host defense peptides, and virulence of Yersinia, we constructed ΔrfaH mutants of Y. pseudotuberculosis IP32953 and Y. pestis KIM6+. Loss of rfaH affected LPS synthesis in both species, resulting in a shorter core oligosaccharide. Susceptibility to polymyxin and the antimicrobial chemokine CCL28 was increased by loss of rfaH in Y. pseudotuberculosis but not in Y. pestis. Transcription of genes in the ddhD-wzz O-antigen gene cluster, but not core oligosaccharide genes, was reduced in ΔrfaH mutants. In addition, mutants with disruptions in specific ddhD-wzz O-antigen cluster genes produced LPS that was indistinguishable from the ΔrfaH mutant. This suggests that both Y. pseudotuberculosis and Y. pestis produce an oligosaccharide core with a single O-antigen unit attached in an RfaH-dependent fashion. Despite enhanced sensitivity to host defense peptides, the Y. pseudotuberculosis ΔrfaH strain was not attenuated in mice, suggesting that rfaH is not required for acute infection.

Molecular bases for multidrug resistance in Yersinia pseudotuberculosis.

The enteropathogen Yersinia pseudotuberculosis causes gastrointestinal infections in humans. Although this species is usually susceptible to antibiotics active against Gram-negative bacteria, we identified three multidrug resistant (MDR) strains of Y. pseudotuberculosis that were isolated from the environment in Russia and from a patient in France. The resistance traits of the two Russian isolates were transferable at high frequencies (≈2×10-1/donor CFU) to Y. pseudotuberculosis. In contrast no transfer of the antibiotic resistances carried by the French strain was observed. Sequencing of the plasmid extracts of the Y. pseudotuberculosis transconjugants for the Russian isolates revealed the presence of conjugative plasmids of the IncN group that carried genes conferring resistance to four to six classes of antibiotics. The French strain harbored a large MDR plasmid of the IncHI2 group that carried resistance genes to six families of antibiotics, and contained a truncated set of transfer genes, accounting for the lack of plasmid transfer. All three Y. pseudotuberculosis plasmids were homologous to MDR plasmids found in various enterobacteria. A phylogenetic analysis showed that the two Russian strain plasmids were closely related to each other and were more distant from the French plasmid. To the best of our knowledge, this is the first molecular characterization of MDR plasmids in Y. pseudotuberculosis. Due to the propensity of this species to acquire exogenous plasmids, the risk of emergence of new MDR Y. pseudotuberculosis isolates should be seriously taken into consideration.

Epigallocatechin gallate inhibits the type III secretion system of Gram-negative enteropathogenic bacteria under model conditions.

Epigallocatechin gallate (EGCG), a major polyphenol in green tea, inhibits the type III secretion system (T3SS) of enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively), Salmonella enterica serovar Typhimurium, and Yersinia pseudotuberculosis. The inhibitory effect causes the inhibition of hemolysis, cell invasion, cell adhesion and apoptosis, which are functions of the type III secretion device. In the case of EPEC, EspB accumulates in the cells. RT-PCR showed that the translation of EspB was not blocked. The transcription of escN, which supplies energy for the injection of the effector factor into the host cells, was also not inhibited. EGCG does not suppress the transcription and translation of T3SS constitutive protein in bacterial cells, but it seems to suppress the normal construction or secretion of T3SS. When Luria-Bertani (LB) medium was used to visualize the EGCG-induced inhibition of T3SS, the inhibitory effect disappeared. The inhibition of T3SS was partially canceled when the T3SS inhibitory potency of EGCG was examined by adding yeast extract, which is a component of LB medium, to DMEM. These results suggest that EGCG probably inhibits secretion by suppressing some metabolic mechanisms of T3SS.

Synthetic Cyclic Peptomers as Type III Secretion System Inhibitors.

Antibiotic-resistant bacteria are an emerging threat to global public health. New classes of antibiotics and tools for antimicrobial discovery are urgently needed. Type III secretion systems (T3SS), which are required by dozens of Gram-negative bacteria for virulence but largely absent from nonpathogenic bacteria, are promising virulence blocker targets. The ability of mammalian cells to recognize the presence of a functional T3SS and trigger NF-κB activation provides a rapid and sensitive method for identifying chemical inhibitors of T3SS activity. In this study, we generated a HEK293 stable cell line expressing green fluorescent protein (GFP) driven by a promoter containing NF-κB enhancer elements to serve as a readout of T3SS function. We identified a family of synthetic cyclic peptide-peptoid hybrid molecules (peptomers) that exhibited dose-dependent inhibition of T3SS effector secretion in Yersinia pseudotuberculosis and Pseudomonas aeruginosa without affecting bacterial growth or motility. Among these inhibitors, EpD-3'N, EpD-1,2N, EpD-1,3'N, EpD-1,2,3'N, and EpD-1,2,4'N exhibited strong inhibitory effects on translocation of the Yersinia YopM effector protein into mammalian cells (>40% translocation inhibition at 7.5 µM) and showed no toxicity to mammalian cells at 240 µM. In addition, EpD-3'N and EpD-1,2,4'N reduced the rounding of HeLa cells caused by the activity of Yersinia effector proteins that target the actin cytoskeleton. In summary, we have discovered a family of novel cyclic peptomers that inhibit the injectisome T3SS but not the flagellar T3SS.

Antimicrobial Use for and Resistance of Zoonotic Bacteria Recovered from Nonhuman Primates.

As a growing threat to human and animal health, antimicrobial resistance (AMR) has become a central public-health topic. Largescale surveillance systems, such as the National Antimicrobial Resistance Monitoring System (NARMS), are now established to monitor and provide guidance regarding AMR, but comprehensive literature on AMR among NHP is sparse. This study provides data regarding current antimicrobial use strategies and the prevalence of AMR in zoonotic bacteria recovered from NHP within biomedical research institutions. We focused on 4 enteric bacteria: Shigella flexneri, Yersinia enterocolitica, Y. pseudotuberculosis, and Campylobacter jejuni. Fifteen veterinarians, 7 biomedical research institutions, and 4 diagnostic laboratories participated, providing susceptibility test results from January 2012 through April 2015. Veterinarians primarily treated cases caused by S. flexneri, Y. enterocolitica, and Y. pseudotuberculosis with enrofloxacin but treated C. jejuni cases with azithromycin and tylosin. All isolates were susceptible to the associated primary antimicrobial but often showed resistance to others. Specifically, S. flexneri isolates frequently were resistant to erythromycin (87.5%), doxycycline (73.7%), and tetracycline (38.3%); Y. enterocolitica isolates to ampicillin (100%) and cefazolin (93.6%); and C. jejuni isolates to methicillin (99.5%) and cephalothin (97.5%). None of the 58 Y. pseudotuber-culosis isolates was resistant to any tested antimicrobial. Notably, resistance patterns were not shared between this study's NHP isolates and human isolates presented by NARMS. Our findings indicate that zoonotic bacteria from NHP diagnostic samples are broadly susceptible to the antimicrobials used to treat the clinical infections. These results can help veterinarians ensure effective antimicrobial therapy and protect staff by minimizing occupational risk.

[INTESTINAL FAILURE AND YERSINIA PSEUDOTUBERCULOSIS TRANSLOCATION IN THE DEVELOPMENTOF EXPERIMENTAL GENERALIZED INFECTION].

THE OBJECTIVE: To determine the value of intestinal failure and translocation of bacteria Y. pseudotuberculosis, and normal intestinal microbiota in the initiation and generalization of infection in experimental pseudotuberculosis in conventional white mice, as well as pathological manifestation of it as a response to the adhesion and colonization of the mucosus membrane by pathogenic bacteria Y. pseudotuberculosis. MATERIALS AND METHODS: Experimental models of pseudotuberculosis in conventional white mice used the pathogenic Y. pseudotuberculosis 147 serotype I strain, containing a calcium-dependence plasmid with a molecular weight of 47 MDa. Cultivation of the pseudotuberculosis pathogen given its psychrophilic was performed on Hottinger agar at a temperature of (4-5) °C. The lactobacilli strain L plantarum 8P-A3 was isolated from a lyophilized commercial probiotic Lactobacterin (manufactured by "NPO Microgen", Russia) and used to obtain native culture supernatant fluid of lactobacilli, the composition of which was detected by gas-liquid chromatography with mass-selective detection. Gentamicin for parenteral administration was manufactured by JSC "Biochemist", Russia. Pathomorphological examination was performed on the 4-6th day of the experiment. Fragments of the small intestine, liver, kidneys, and lungs from dead animals were chosen for examination. Tissues were fixed in 10% neutral formalin, dehydrated in isopropanol and embedded in paraffin. Preparations were stained with Ehrlich hematoxylin and eosin, examined on the microscope "Mikmed-2" (JSC "LOMO", Russia) under magnification x 200-x1000. Statistical processing of the experimental results was carried out according to the method of Kerber in modification of I.P. Ashmarin and A.A. Vorobyov. RESULTS: The role of intestinal failure and translocation of bacteria Y. pseudotuberculosis, and normal intestinal microbiota in the initiation and generalization of infection in animals has been found. It has been proved that the oral administration of supernatant fluid containing microbial metabolites to animals as intramuscular administration of gentamicin equally prevent the development of generalized pseudotuberculosis and intensity of pathomorphological changes in the intestine and other organs of animals. CONCLUSION: Metabolites of the probiotic lactobacilli strain L plantarum 8P-A3 jugulate the development of pseudotuberculosis at an early stage of the pathological process in experimental animals infected with pathogen Y pseudotuberculosis, not only causing the preservation of the colonization resistance of the intestinal mucosa that prevents the adhesion and colonization of the pathogen, but also through their antimicrobial impact on the dissiminated pseudotuberculosis patho-gen bacteria in animals.

CD8(+) T cells specific to a single Yersinia pseudotuberculosis epitope restrict bacterial replication in the liver but fail to provide sterilizing immunity.

CD8(+) T cells use contact-dependent cytolysis of target cells to protect the host against intracellular pathogens. We have previously shown that CD8(+) T cells and perforin are required to protect against the extracellular pathogen Yersinia pseudotuberculosis. Here we establish an experimental system where CD8(+) T cells specific to a single model antigen are the only memory response present at time of challenge. Using mice immunized with a vaccine strain of Listeria monocytogenes that expresses secreted ovalbumin (Lm-OVA), we show that OVA-specific CD8(+) T cells are generated and provide limited protection against challenge with virulent OVA(+)Y. pseudotuberculosis. Perforin expression by OVA-specific CD8(+) T cells was required, as Lm-OVA-immunized perforin-deficient mice showed higher bacterial burden as compared to Lm-OVA-immunized perforin-sufficient mice. Surprisingly, antigen-specific T cell protection waned over time, as Lm-OVA-immune mice eventually succumbed to Yersinia infection. Kinetic analysis of infection in mice with and without OVA-specific CD8(+) T cells revealed that bacterial numbers increased sharply in OVA-naïve mice until death, while OVA-immune mice held bacterial burden to a lower level throughout the duration of illness until death. Clonal analysis of bacterial populations in OVA-naïve and OVA-immune mice at distinct time points revealed equivalent and severe bottle-neck effects for bacteria in both sets of mice immediately after intravenous challenge, demonstrating a dominant role for other aspects of the immune system regardless of CD8(+) T cell status. These studies indicate that CD8(+) T cells against a single antigen can restrict Y. pseudotuberculosis colonization in a perforin-dependent manner, but ultimately are insufficient in their ability to provide sterilizing immunity and protect against death.

Pyogenic vertebral osteomyelitis caused by Yersinia pseudotuberculosis.

Septic vertebral osteomyelitis caused by Yersinia pseudotuberculosis is extremely rare. We report the first case of pyogenic vertebral osteomyelitis due to Y. pseudotuberculosis. A 65-year-old man presented with low back pain and fever. He had a history of psoriasis vulgaris treated with adalimumab. The patient reported drinking adequate amounts of well water daily. Clinical examination revealed no neurological deficit of the lower limbs. Laboratory findings included increased inflammatory reactions. Magnetic resonance imaging showed diffuse changes in intensity at the T10 and L3 vertebrae, and multiple nodular lesions in the thoracic, lumbar, sacral, and iliac regions. We performed an open biopsy. Y. pseudotuberculosis was cultured from both the T10 and L3 vertebrae lesions. Y. pseudotuberculosis is sensitive to almost all antibiotics. After initiating antibiotic treatment, the laboratory parameters normalized and the patient recovered completely, without any neurological deficits. To our knowledge, our report represents the first case of pyogenic vertebral osteomyelitis due to Y. pseudotuberculosis.

Bacteriomimetic invasin-functionalized nanocarriers for intracellular delivery.

Intracellular bacteria invade mammalian cells to establish an infectious niche. The current work models adhesion and subsequent internalization strategy of pathogenic bacteria into mammalian cells to design a bacteriomimetic bioinvasive delivery system. We report on the surface functionalization of liposomes with a C-terminal fragment of invasin (InvA497), an invasion factor in the outer membrane of Yersinia pseudotuberculosis. InvA497-functionalized liposomes adhere to mammalian epithelial HEp-2 cell line at different infection stages with a significantly higher efficiency than liposomes functionalized with bovine serum albumin. Covalent attachment of InvA497 results in higher cellular adhesion than liposomes with physically adsorbed InvA497 with non-specific surface protein alignment. Uptake studies in HEp-2 cells indicate active internalization of InvA497-functionalized liposomes via ß1-integrin receptor-mediated uptake mechanism mimicking the natural invasion strategy of Y. pseudotuberculosis. Uptake studies in Caco-2 cells at different polarization states demonstrate specific targeting of the InvA497-functionalized liposomes to less polarized cells reflecting the status of inflamed cells. Moreover, when loaded with the anti-infective agent gentamicin and applied to HEp-2 cells infected with Y. pseudotuberculosis, InvA497-functionalized liposomes are able to significantly reduce the infection load relative to non-functionalized drug-loaded liposomes. This indicates a promising application of such a bacteriomimetic system for drug delivery to intracellular compartments.

[CHARACTERISTICS OF FORMATION, INHIBITION AND DESTRUCTION OF YERSINIA PSEUDOTUBERCULOSIS BIOFILMS FORMING ON ABIOTIC SURFACES].

AIM: Detection of conditions of Yersinia pseudotuberculosis biofilm formation, their quantitative testing. MATERIALS AND METHODS: Y. pseudotuberculosis strains, nutrient media, standard 96-well polystyrene plates, crystal violet dye as well as bacteriologic, spectrophotometric, statistical methods were used. RESULTS: All the studied Y pseudotuberculosis strains formed a well expressed biofilm on abiotic surface during cultivation of bacteria in 200 µl of a plate well at a temperature of 20-22°C for 4-7 days. Bacteria CFU number in biofilm reduced by day 10 of incubation. DNAse I was found to inhibit biofilm formation, and also partially destroyed mature Y. pseudotuberculosis biofilm. The presence of DNA in extra-cellular matrix of biofilm was shown. CONCLUSION: An ability of Y. pseudotuberculosis to form biofilm on abiotic surface was established. The conditions of biofilm formation were determined. Inhibiting effect of DNAse I on Y. pseudotuberculosis was shown.

Design, synthesis and evaluation of novel polypharmacological antichlamydial agents.

Discovery of new polypharmacological antibacterial agents with multiple modes of actions can be an alternative to combination therapy and also a possibility to slow development of antibiotic resistance. In support to this hypothesis, we synthesized 16 compounds by combining the pharmacophores of Chlamydia trachomatis inhibitors and inhibitors of type III secretion (T3S) in gram-negative bacteria. In this study we have developed salicylidene acylhydrazide sulfonamides (11c &11d) as new antichlamydial agents that also inhibit T3S in Yersinia pseudotuberculosis.