Intracellular Survival Assays Research Articles

Diagnostic value of lncRNAs LINC00152 and LARS2-AS1 and their regulatory roles in macrophage immune response in tuberculosis

ObjectivesTo determine the usefulness of LINC00152 and LARS2-AS1 as potential biomarkers for latent tuberculosis (LTB) and active tuberculosis (ATB), as well as their effect on Mycobacterium (Mtb) infection. MethodsThe expression levels of LINC00152 and LARS2-AS1 in the health, patients with LTB and ATB were detected by qRT-PCR. The ROC curves were constructed to show their potential as biomarkers. The intracellular survival assays for Mtb and the levels of immune-related cytokines were determined to discover the effect of LINC00152 and LARS2-AS1 on Mtb infection. The relationships of miR-485-5p with LINC00152 and LARS2-AS1 were explored. ResultsLINC00152 and LARS2-AS1 levels were significantly elevated in patients with ATB and LTB, and Mtb-infected macrophages. LINC00152 and LARS2-AS1 can distinguish the LTB from the health and ATB from LTB. LARS2-AS1 and LINC00152 knock-down reduced the intracellular Mtb survival and induced cellular immune response after Mtb challenge. miR-485-5p was a targeting miRNA for LINC00152 and LARS2-AS1. ConclusionsLINC00152 and LARS2-AS1 can be considered as potential biomarkers for tuberculosis disease. LINC00152 and LARS2-AS1 have anti-Mtb effects.

CheV enhances the virulence of Salmonella Enteritidis, and the Chev-deleted Salmonella vaccine provides immunity in mice

BackgroundSalmonella enteritidis (SE) is a major zoonotic pathogen and causes infections in a variety of hosts. The development of novel vaccines for SE is necessary to eradicate this pathogen. Genetically engineered attenuated live vaccines are more immunogenic and safer. Thus, to develop a live attenuated Salmonella vaccine, we constructed a cheV gene deletion strain of SE (named ΔcheV) and investigated the role of cheV in the virulence of SE. First, the ability to resist environmental stress in vitro, biofilm formation capacity, drug resistance and motility of ΔcheV were analyzed. Secondly, the bacterial adhesion, invasion, intracellular survival assays were performed by cell model. Using a mouse infection model, an in vivo virulence assessment was conducted. To further evaluate the mechanisms implicated by the reduced virulence, qPCR analysis was utilized to examine the expression of the strain’s major virulence genes. Finally, the immune protection rate of ΔcheV was evaluated using a mouse model.ResultsCompared to C50336, the ΔcheV had significantly reduced survival ability under acidic, alkaline and thermal stress conditions, but there was no significant difference in survival under oxidative stress conditions. There was also no significant change in biofilm formation ability, drug resistance and motility. It was found that the adhesion ability of ΔcheV to Caco-2 cells remained unchanged, but the invasion ability and survival rate in RAW264.7 cells were significantly reduced. The challenge assay results showed that the LD50 values of C50336 and ΔcheV were 6.3 × 105 CFU and 1.25 × 107 CFU, respectively. After the deletion of the cheV gene, the expression levels of fimD, flgG, csgA, csgD, hflK, lrp, sipA, sipB, pipB, invH, mgtC, sodC, rfbH, xthA and mrr1 genes were significantly reduced. The live attenuated ΔcheV provided 100% protection in mice against SE infection.ConclusionAll the results confirmed that the deletion of the cheV gene reduces the virulence of SE and provides significant immune protection in mice, indicating that ΔcheV could be potential candidates to be explored as live-attenuated vaccines.

Antibiotic resistance, pathotypes, and pathogen-host interactions in Escherichia coli from hospital wastewater in Bulawayo, Zimbabwe.

This study aimed to characterise E. coli strains isolated from hospital wastewater effluent in Bulawayo, Zimbabwe, using both molecular and cytological approaches. Wastewater samples were aseptically collected from the sewerage mains of a major public referral hospital in Bulawayo province weekly for one month. A total of 94 isolates were isolated and confirmed as E. coli through biotyping and PCR targeting the uidA housekeeping gene. A total of 7 genes (eagg, eaeA, stx, flicH7, ipaH, lt, and st genes) coding for virulence in diarrheagenic E. coli were targeted. Antibiotic susceptibility of E. coli was determined against a panel of 12 antibiotics through the disk diffusion assay. The infectivity status of the observed pathotypes was investigated using HeLa cells through adherence, invasion, and intracellular assay. None of the 94 isolates tested positive for the ipaH and flicH7genes. However, 48 (53.3%) isolates were enterotoxigenic E. coli (ETEC) (lt gene positive), 2 (2.13%) isolates were enteroaggregative E. coli (EAEC) (eagg gene), and 1 (1.06%) isolate was enterohaemorrhagic E. coli (EHEC) (stx and eaeA). A high level of sensitivity was observed in E. coli against ertapenem (98.9%), and Azithromycin (75.5%). The highest resistance was against ampicillin (92.6%) and sulphamethoxazole-trimethoprim (90.4%). Seventy-nine (84%) E. coli isolates exhibited multidrug resistance. The infectivity study results indicated that environmentally isolated pathotypes were as infective as the clinically isolated pathotypes for all three parameters. No adherent cells were observed using ETEC, and no cells were observed in the intracellular survival assay using EAEC. This study revealed that hospital wastewater is a hotspot for pathogenic E. coli and that the environmentally isolated pathotypes maintained their ability to colonise and infect mammalian cells.

Investigation of Drug ResistantAcinetobacter baumannii Virulence Factors Implicated in Intracellular Survival Within Differentiated THP-1 Cells

Abstract Introduction/Objective Acinetobacter baumannii is a nosocomial opportunistic pathogen that is usually associated with mucosal infections such as pneumonia, urinary tract infections or sepsis and less commonly with more severe illnesses such as meningitis, wound infection, or necrotizing fasciitis. Of late, A. baumanniihas been flagged by the CDC as an urgent threat due to multidrug-resistant properties it has acquired, making it a major concern to public health. Due to a sparsity of information on the virulence factors of immune evasion, it has been difficult to understand how this bacterium is able to gain a foothold and cause severe infections. We sought to study how necrotizing fasciitis-associated strains of A. baumannii interact with human macrophages to assess their capabilities in evasion of the host immune response. Methods/Case Report To this end, extensively drug resistant A. baumannii was isolated at two timepoints (strains NFAb-1 and NFAb-2) from a patient over the course of a lethal necrotizing fasciitis infection that led to sepsis. The intracellular survival of these two strains was compared with each other, as well as to the type strains, ATCC 19606 and ATCC 17978, within a human macrophage-differentiated monocyte cell line, THP-1. Survival studies were complemented with quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) to investigate expression of potential bacterial virulence factors, Catalase E (KatE), Phospholipase C (PLC), Outer membrane protein A (ompA), and Pilus A (PilA), that could contribute to immune evasion. Results (if a Case Study enter NA) The intracellular survival assay demonstrated enhanced survival and bacterial growth of NFAb-1 and NFAb-2 within macrophages compared to ATCC 19606 and 17978. Examining gene expression, KatE, PLC, and OmpA didn’t show any significant differences between the bacterial strains, while the PilA gene had much higher expression in NFAb-1 and NFAb-2 compared to ATCC 19606 and 17978. Conclusion This data establishes these nectrotizing fasciitis-causing A. baumannii strains are more likely to sruvivie a macrophage-mediatied immune response and expression of pilus-assocated genes may play a role in these interactions. our studies demonstrate a need to further investigate A. baumannii mechanisms of virulence to understand its ability to evade the immune response in hopes of understanding and combating infections.

P349 Relationship between genotypes and in vitro pathogenic behavior of Uruguayan strains of Cryptococcus spp.

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjectiveContribute to the knowledge of the relationship between genetic and pathogenic characteristics of meningeal cryptococcosis-producing strains from Uruguay.MethodsWe have included a total of 21 isolates of Cryptococcus spp from human cases of meningoencephalitis. We start from the general strain collection of the mycology laboratory of the department of parasitology and mycology, medical school, University of the Republic. The strain was sectioned taking into account the phenotypic diversity and genetic diversity through a previous analysis by PCR fingerprinting, for study by MLST.MLST was performed according to the protocol available at: http://mlst.mycologylab.org/defaultinfo.aspx?Page=CryptoHome.Infection, intracellular survival, and cell proliferation assays were performed. The murine macrophage cell line J774 (ATCC TIB 67) was used.ResultsOf the 21 strains studied, in 13 we obtained an ST that had been previously reported (9 C. neoformans complex and 4 C. gattii complex). For the remaining 8 strains, we obtained new allelic combinations to which new STs were assigned, which were deposited in the MLST database for Cryptococcus spp. (http://mlst.mycologylab.org) (Table 1).All strains were phagocytosed and all were able to persist within macrophages at 24 h post-infection, although there was great variability between strains (Table 1). In most of the strains, the control of macrophages prevailed through cell lysis mechanisms over Cryptococcus spp, with one proliferation index (PI) ˂1.0. In the proliferation assay, only 4 strains managed to proliferate within the macrophages, 3 of them correspond to C. gattii complex, genotypes VGI and VGII. The 4th strain that achieved proliferation was corresponding to the VNI genotype.ConclusionWe obtained a clear differentiation of in vitro behavior between the strains corresponding to C. neoformans complex and C. gattii complex. The strains belonging to C. gattii complex presented less phagocytosis and greater persistence and intracellular proliferation.

Supervivencia intracelular de cepas de Mycobacterium bovis de alta y baja frecuencia probado en un modelo de macrófagos bovinos

Bovine tuberculosis is a disease caused by Mycobacterium bovis that affects cattle and other species, including humans. Mycobacterium bovis resides mainly in macrophages, so bacilli survival within macrophages is related to virulence. Isolation and strain identification are important for disease control. However, little is known about virulence of the circulating strains in cattle populations. Therefore, the aim of this study was to compare the intracellular survival of Mycobacterium bovis strains with high and low frequency genotypes in cattle in Mexico. Four high frequency genotypes and four low frequency genotypes were identified and subjected to intracellular survival assays in bovine macrophages. Results showed that the phagocytosis proportion was approximately 63 % for all strains. There were no significant differences in the average Colony Forming Units (CFUs) in phagocytosis and survival between the high and low frequency groups; however, when the CFU average of phagocytosis was compared with the survival, significant differences were found in both groups. In intracellular growth, a significant difference was observed between low and high frequency strains, and between low frequency strains. Finally, the intracellular growth average of the groups was analyzed showing no significant difference. These results suggest that the frequency of the genotype in cattle population is not related to the intracellular survival and the virulence of the M. bovis strains.

Antibiotic resistance and virulence genes in nisin-resistant Enterococcus faecalis isolated from raw buffalo milk modulate the innate functions of rat macrophages.

To elucidate the antibiotic resistance and virulence genes of nisin-resistant Enterococcus faecalis isolated from raw buffalo milk and to study the effect of nisin-sensitive and -resistant E. faecalis on the innate immunity of rats. Slanetz-Bartley agar plates containing nisin were used to isolate nisin-resistant E. faecalis. The virulence factors were ascertained using quantitative real-time polymerase chain reaction. Cell viability, phagocytosis, intracellular survival and enzyme assays were performed to investigate the interaction of E. faecalis with rat macrophages. Nisin-resistant E. faecalis was less prone to phagocytosis and survived longer inside the macrophages, due to reduced production of reactive oxygen species and nitric oxide. The viability and activation of macrophages was also reduced in the presence of resistant E. faecalis, as observed by enhanced lactate dehydrogenase production and reduced β-galactosidase. Nisin-resistant E. faecalis and its virulence factors were reported in raw buffalo milk. This study shows that nisin-resistant variants exhibited cross resistance to antibiotics and suppressed the innate immune responses of rats by directly affecting macrophage activity. This study elucidated the contamination of raw buffalo milk by nisin-resistant E. faecalis, which may pose food safety risk.

Streptococcus pneumoniae resists intracellular killing by olfactory ensheathing cells but not by microglia.

Olfactory ensheathing cells (OECs) are a type of specialized glial cell currently considered as having a double function in the nervous system: one regenerative, and another immune. Streptococcus pneumoniae is a major agent of severe infections in humans, including meningitis. It is commonly found in the nasopharynx of asymptomatic carriers, and, under certain still unknown conditions, can invade the brain. We evaluated whether pneumococcal cells recovered from lysed OECs and microglia are able to survive by manipulating the host cell activation. An intracellular-survival assay of S. pneumoniae in OECs showed a significant number of bacterial CFU recovered after 3 h of infection. In contrast, microglia assays resulted in a reduced number of CFU. Electron-microscopy analysis revealed a large number of pneumococci with apparently intact morphology. However, microglia cells showed endocytic vesicles containing only bacterial cell debris. Infection of OEC cultures resulted in continuous NF-κB activation. The IFN-γ-induced increase of iNOS expression was reversed in infected OECs. OECs are susceptible to S. pneumoniae infection, which can suppress their cytotoxic mechanisms in order to survive. We suggest that, in contrast to microglia, OECs might serve as safe targets for pneumococci, providing a more stable environment for evasion of the immune system.

Evaluation of ompA and pgtE genes in determining pathogenicity in Salmonella enterica serovar Enteritidis

Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) is a major causative agent of gastroenteritis in humans. This important food-borne pathogen also colonises the intestinal tracts of poultry and can spread systemically, especially in chickens. To identify the S. Enteritidis virulence genes involved in infection and colonisation of chickens, chromosomal deletion mutants of the ompA and pgtE genes, which encode essential components of omptins, were constructed.There were no significant differences between the wild-type and ompA and pgtE mutants in a series of in vitro assays, including an intracellular survival assay, survival in specific-pathogen-free (SPF) chicken serum, and in vitro competition assays. In contrast, in vivo competition assays revealed that ompA and pgtE mutants underwent attenuated growth in liver, cardiac blood, spleen, lung, and kidney compared to a wild-type strain (CVCC3378). When tested in SPF chickens, ompA or pgtE gene inactivation substantially reduced organ colonisation and delayed systemic infection compared with the wild-type strain. Colonisation was restored in S. Enteritidis mutants by reintroduction of the whole ompA or pgtE gene with the native promoters. The results of this study demonstrate that ompA and pgtE play an important role in the pathogenesis of S. Enteritidis and its ability to infect chickens.

The role of genomic islands in Escherichia coli K1 interactions with intestinal and kidney epithelial cells

The completion of Escherichia coli K1 genome has identified several genomic islands that are present in meningitis-causing E. coli RS218 but absent in the non-pathogenic E. coli MG1655. In this study, the role of various genomic islands in E. coli K1 interactions with intestinal epithelial cells (Caco-2) and kidney epithelial cells (MA104) was determined. Using association assays, invasion assays, and intracellular survival assays, the findings revealed that the genomic island deletion mutants of RS218 related to P fimbriae, S fimbriae, F17-like fimbriae, non-fimbrial adhesins, Hek and hemagglutinin, protein secretion system (T1SS for hemolysin; T2SS; T5SS for antigen 43), Iro system and hmu system), invasins (CNF1, IbeA), toxins (α-hemolysin), K1 capsule biosynthesis, metabolism (d-serine catabolism, dihydroxyacetone, glycerol, and glyoxylate metabolism), prophage genes, showed reduced interactions with both cell types. Next, we determined the role of various genomic islands in E. coli K1 resistance to serum. When exposed to the normal human serum, the viability of the genomic island deletion mutants related to adhesins such as S fimbriae, P fimbriae, F17-like fimbriae, non-fimbrial adhesins, Hek and hemagglutinin, antigen 43 and T5SS for antigen 43, T2SS, and T1SS for hemolysin, Iro system and hmu system, prophage genes, metabolism (sugar metabolism and d-serine catabolism), K1 capsule biosynthesis, and invasins such as CNF1 was affected, suggesting their role in bacteremia. The characterization of these genomic islands should reveal mechanisms of E. coli K1 pathogenicity that could be of value as therapeutic targets.

Altered Proteome of Burkholderia pseudomallei Colony Variants Induced by Exposure to Human Lung Epithelial Cells.

Burkholderia pseudomallei primary diagnostic cultures demonstrate colony morphology variation associated with expression of virulence and adaptation proteins. This study aims to examine the ability of B. pseudomallei colony variants (wild type [WT] and small colony variant [SCV]) to survive and replicate intracellularly in A549 cells and to identify the alterations in the protein expression of these variants, post-exposure to the A549 cells. Intracellular survival and cytotoxicity assays were performed followed by proteomics analysis using two-dimensional gel electrophoresis. B. pseudomallei SCV survive longer than the WT. During post-exposure, among 259 and 260 protein spots of SCV and WT, respectively, 19 were differentially expressed. Among SCV post-exposure up-regulated proteins, glyceraldehyde 3-phosphate dehydrogenase, fructose-bisphosphate aldolase (CbbA) and betaine aldehyde dehydrogenase were associated with adhesion and virulence. Among the down-regulated proteins, enolase (Eno) is implicated in adhesion and virulence. Additionally, post-exposure expression profiles of both variants were compared with pre-exposure. In WT pre- vs post-exposure, 36 proteins were differentially expressed. Of the up-regulated proteins, translocator protein, Eno, nucleoside diphosphate kinase (Ndk), ferritin Dps-family DNA binding protein and peptidyl-prolyl cis-trans isomerase B were implicated in invasion and virulence. In SCV pre- vs post-exposure, 27 proteins were differentially expressed. Among the up-regulated proteins, flagellin, Eno, CbbA, Ndk and phenylacetate-coenzyme A ligase have similarly been implicated in adhesion, invasion. Protein profiles differences post-exposure provide insights into association between morphotypic and phenotypic characteristics of colony variants, strengthening the role of B. pseudomallei morphotypes in pathogenesis of melioidosis.

Possible Role of Toll‐like Receptor‐2 in the Intracellular Survival of Staphylococcus aureus in Murine Peritoneal Macrophages: Involvement of Cytokines and Anti‐Oxidant Enzymes

Effects of blocking toll-like receptor-2 (TLR-2) on the survival of Staphylococcus aureus (S. aureus) and cytokine production in peritoneal macrophages of Swiss albino mice were analysed. Macrophages were infected with S. aureus in the presence and absence of anti-TLR-2 antibody. Tumour necrosis factor-α (TNF-α) interleukin-6 (IL-6), interferon-gamma (IFN-γ), interleukin-1β (IL-1β), interleukin-12 (IL-12) and interleukin-10 (IL-10) concentrations were measured. Expressions of TLR-2, NF-κB, MyD 88 were analysed by Western Blot. Expression of TLR-2 was increased in S. aureus-infected macrophages with respect to control and was MyD 88 independent. TLR2 blocking significantly reduced TNF-α, IL-6, IL-1β and IL-10 and increased IFN-γ and IL-12 production. Decreased catalase activity and increased superoxide dismutase (SOD) by S. aureus with concomitant increase in H2 O2 and nitric oxide (NO) were observed in the case of prior TLR-2 blocking. To understand whether catalase contributing in the intracellular survival, was of bacterial origin or not, 3-amino, 1, 2, 4-triazole (ATZ) was used to inhibit specifically macrophage-derived catalase. Catalase enzyme activity from the whole staphylococcal cells in the presence of ATZ suggested that the released catalase were of extracellular origin. From the intracellular survival assay, it was evident that pretreatment of macrophages with ATZ reduces the bacterial burden in macrophages when infected with the recovered bacteria only from the anti-TLR-2 antibody-treated macrophages after phagocytosis. Catalase protein expression from the whole staphylococcal cells recovered after phagocytosis also indicated the catalase release from S. aureus. Capturing of S. aureus via TLR-2 induces inflammatory reactions through activation of NF-κB-signalling pathways which was MyD88-independent.

Evaluation of antibacterial and cytotoxic activity of Artemisia nilagirica and Murraya koenigii leaf extracts against mycobacteria and macrophages

BackgroundArtemisia nilagirica (Asteraceae) and Murraya koenigii (Rutaceae) are widely distributed in eastern region of India. Leaves of Artemisia nilagirica plant are used to treat cold and cough by the local tribal population in east India. Murraya koenigii is an edible plant previously reported to have an antibacterial activity. Pathogenic strains of mycobacteria are resistant to most of the conventional antibiotics. Therefore, it is imperative to identify novel antimycobacterial molecules to treat mycobacterial infection.MethodsIn this study, ethanol, petroleum ether and water extracts of Artemisia nilagirica and Murraya koenigii were tested for antibacterial activity against Mycobacterium smegmatis and Mycobacterium bovis BCG in synergy with first line anti-tuberculosis (TB) drugs, and for cytotoxic activities on mouse macrophage RAW264.7 cells. Antibacterial activity was determined by colony forming unit (CFU) assay. Intracellular survival assay was performed by infecting RAW264.7 cells with M. smegmatis before and after treatment with plant extracts. Cytotoxity was checked by MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] assay. Genotoxicity was studied by DAPI staining and COMET assay using mouse macrophage RAW264.7 cell line. Cell apoptosis was checked by Annexin-V/FITC dual staining method. Reactive oxygen species and nitric oxide production was checked by DCFH staining and Griess reagent, respectively.ResultsEthanol extracts of A. nilagirica (IC50 300 μg/ml) and M. koenigii (IC50 400 μg/ml) were found to be more effective against Mycobacterium smegmatis as compared to petroleum ether and water extracts. M. koenigii extract showed maximum activity against M. bovis BCG in combination with a first line anti-TB drug rifampicin. M. koenigii leaf extract also exerted more cytototoxic (IC50 20 μg/ml), genotoxic and apoptosis in mouse macrophage RAW 264.7 cell line. Treatment of mouse macrophages with A. nilagirica extract increased intracellular killing of M. smegmatis by inducing production of reactive oxygen species and nitric oxide.ConclusionsEthanol extracts of A. nilagirica and M. koenigii were found to be more effective against mycobacteria. As compared to A. nilagirica, M. koenigii ethanol extract exhibited significant synergistic antibacterial activity against M. smegmatis and M. bovis BCG in combination with anti-tuberculosis drug rifampicin, and also showed increased cytotoxicity, DNA damage and apoptosis in mouse macrophages.

Yeast synthetic biology platform generates novel chemical structures as scaffolds for drug discovery.

Synthetic biology has been heralded as a new bioengineering platform for the production of bulk and specialty chemicals, drugs, and fuels. Here, we report for the first time a series of 74 novel compounds produced using a combinatorial genetics approach in baker’s yeast. Based on the concept of “coevolution” with target proteins in an intracellular primary survival assay, the identified, mostly scaffold-sized (200–350 MW) compounds, which displayed excellent biological activity, can be considered as prevalidated hits. Of the molecules found, >75% have not been described previously; 20% of the compounds exhibit novel scaffolds. Their structural and physicochemical properties comply with established rules of drug- and fragment-likeness and exhibit increased structural complexities compared to synthetically produced fragments. In summary, the synthetic biology approach described here represents a completely new, complementary strategy for hit and early lead identification that can be easily integrated into the existing drug discovery process.

RfaH Promotes the Ability of the Avian Pathogenic Escherichia coli O2 Strain E058 To Cause Avian Colibacillosis

Avian pathogenic Escherichia coli (APEC) infection causes avian colibacillosis, which refers to any localized or systemic infection, such as acute fatal septicemia or subacute pericarditis and airsacculitis. The RfaH transcriptional regulator in E. coli is known to regulate a number of phenotypic traits. The direct effect of RfaH on the virulence of APEC has not been investigated yet. Our results showed that the inactivation of rfaH significantly decreased the virulence of APEC E058. The attenuation was assessed by in vivo and in vitro assays, including chicken infection assays, an ingestion and intracellular survival assay, and a bactericidal assay with serum complement. The virulence phenotype was restored to resemble that of the wild type by complementation of the rfaH gene in trans. The results of the quantitative real-time reverse transcription-PCR (qRT-PCR) analysis and animal system infection experiments indicated that the deletion of rfaH correlated with decreased virulence of the APEC E058 strain.

Thermostable Hexameric Form of Eis (Rv2416c) Protein of M. tuberculosis Plays an Important Role for Enhanced Intracellular Survival within Macrophages

Eis protein is reported to enhance the intracellular survival of Mycobacterium tuberculosis in human macrophages. Eis protein is not only known to skew away the immunity by disturbing the protective TH1 response, but aminoglycoside acetyltransferase activity of Eis is reported to regulate autophagy, inflammation and cell death. Here we have gained insight into the structure-function properties of Eis. Eis protein is a hexameric αβ protein. Although urea and guanidinium hydrochloride (GdmCl) was found to induce one-step unfolding of Eis but size exclusion chromatography showed that GdmCl treated Eis maintained its hexameric form. SDS-PAGE assay confirmed that hexameric form of Eis is partially stable to SDS and converts into trimers and monomers. Out of these three forms, aminoglycoside acetyltransferase activity is found to be associated only with hexamers. The Tm of Eis was found to be ∼75°C. Aminoglycoside acetyltransferase Eis demonstrated remarkable heat stability retaining >80% of their activity at 70°C which falls down to ∼50% at 75°C and is completely inactive at 80°C. Further, intracellular survival assay with heated samples of M. smegmatis harboring eis gene of M. tuberculosis H37Rv demonstrated a possible role for the thermostability associated with Eis protein in the enhanced intracellular survival within macrophages. In sum, these data reveal that only hexameric form of Eis has a thermostable aminoglycoside acetyltransferase activity. This is the first report showing the thermostability associated with aminoglycoside acetyltransferase activity of Eis protein being one of the essential features for the execution of its biological role.

Persistent survival of luminescent mycobacterium avium subspecies paratuberculosis in peripheral blood monocytes from both crohn's disease and healthy controls is associated with differential cytokine response

IntroductionAbnormal intestinal macrophage function which could allow persistence of candidate bacterial triggering agents such as E coli and Mycobacterium avium subspecies paratuberculosis (MAP), has been implicated in the pathogenesis of...

In vitro markers for virulence in Yersinia ruckeri

In this study, different traits that have been associated with bacterial virulence were studied in Yersinia ruckeri. Two isolates that had been shown to cause disease and mortality in experimentally infected rainbow trout were compared with five avirulent isolates. Both virulent isolates showed high adhesion to gill and intestinal mucus of rainbow trout, whereas the majority of non-virulent strains demonstrated significantly lower adhesion. A decrease in adherence capability following bacterial treatment with sodium metaperiodate and proteolytic enzymes suggested the involvement of carbohydrates and proteins. All strains were able to adhere to and invade chinook salmon embryo cell line (CHSE-214), fathead minnow epithelial cell line (FHM) and rainbow trout liver cell line (R1). One non-virulent strain was highly adhesive and invasive in the three cell lines, whereas the virulent strains showed moderate adhesive and invasive capacity. The internalization of several isolates was inhibited by colchicine and cytochalasin-D, suggesting that microtubules and microfilaments play a role. For all strains, intracellular survival assays showed a decrease of viable bacteria in the cells 6 h after inoculation, suggesting that Y. ruckeri is not able to multiply or survive inside cultured cells. Analysis of the susceptibility to the bactericidal effect of rainbow trout serum demonstrated that virulent Y. ruckeri strains were serum resistant, whereas non-virulent strains were generally serum sensitive.

Atypical Roles for Campylobacter jejuni Amino Acid ATP Binding Cassette Transporter Components PaqP and PaqQ in Bacterial Stress Tolerance and Pathogen-Host Cell Dynamics

Campylobacter jejuni is a human pathogen causing severe diarrheal disease; however, our understanding of the survival of C. jejuni during disease and transmission remains limited. Amino acid ATP binding cassette (AA-ABC) transporters in C. jejuni have been proposed as important pathogenesis factors. We have investigated a novel AA-ABC transporter system, encoded by cj0467 to cj0469, by generating targeted deletions of cj0467 (the membrane transport component) and cj0469 (the ATPase component) in C. jejuni 81-176. The analyses described here have led us to designate these genes paqP and paqQ, respectively (pathogenesis-associated glutamine [q] ABC transporter permease [P] and ATPase [Q]). We found that loss of either component resulted in amino acid uptake defects, most notably diminished glutamine uptake. Altered resistance to a series of environmental and in vivo stresses was also observed: both mutants were hyperresistant to aerobic and organic peroxide stress, and while the DeltapaqP mutant was also hyperresistant to heat and osmotic shock, the DeltapaqQ mutant was more susceptible than the wild type to the latter two stresses. The DeltapaqP and DeltapaqQ mutants also displayed a surprising but statistically significant increase in recovery from macrophages and epithelial cells in short-term intracellular survival assays. Annexin V, 4',6-diamidino-2-phenylindole (DAPI), and Western blot analyses revealed that macrophages infected with the DeltapaqP or DeltapaqQ mutant exhibited transient but significant decreases in cell death and extracellular signal-regulated kinase-mitogen-activated protein kinase activation compared to levels in wild-type-infected cells. The DeltapaqP mutant was not defective in either short-term or longer-term mouse colonization, consistent with its increased stress survival and diminished host cell damage phenotypes. Collectively, these results demonstrate a unique correlation of an AA-ABC transporter with bacterial stress tolerances and host cell responses to pathogen infection.

Transcriptome studies on Streptococcus pneumoniae, illustration of early response genes to THP-1 human macrophages

Pathogen–host interaction plays an essential role in pathogenicity. In this study, we investigated transcriptomes of one Streptococcus pneumoniae TIGR4-derived unencapsulated strain upon exposure to THP-1 human macrophage-like cells for 0.5 h, 1 h and 3 h, respectively. Expression of most genes was up-regulated and the changes of selected genes were validated by qRT-PCR. To characterize the functions of the identified genes, one locus of genes (SP1057–SP1063) was deleted in TIGR4 by insertion replacement mutagenesis. Compared to the wild-type strain, the constructed mutant exhibited lower binding and internalization activities to the THP-1 macrophages at early incubation time periods (0.5 h and/or 1 h) but not at 3 h. However, no change was observed in the intracellular survival assays. These data indicate that the SP1057–SP1063 locus is involved in the early stage of interaction with host macrophages. Further sequence and PCR analyses suggest that the SP1057–SP1063 locus was acquired by lateral transfer.