Dual Inhibition of Vacuolar-ATPase and TMPRSS2 Is Required for Complete Blockade of SARS-CoV-2 Entry into Cells.

An essential step in the infection life cycle of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the proteolytic activation of the viral spike (S) protein, which enables membrane fusion and entry into the host cell. Two distinct classes of host proteases have been implicated in the S protein activation step: cell-surface serine proteases, such as the cell-surface transmembrane protease, serine 2 (TMPRSS2), and endosomal cathepsins, leading to entry through either the cell-surface route or the endosomal route, respectively. In cells expressing TMPRSS2, inhibiting endosomal proteases using nonspecific cathepsin inhibitors such as E64d or lysosomotropic compounds such as hydroxychloroquine fails to prevent viral entry, suggesting that the endosomal route of entry is unimportant; however, mechanism-based toxicities and poor efficacy of these compounds confound our understanding of the importance of the endosomal route of entry. Here, to identify better pharmacological agents to elucidate the role of the endosomal route of entry, we profiled a panel of molecules identified through a high-throughput screen that inhibit endosomal pH and/or maturation through different mechanisms. Among the three distinct classes of inhibitors, we found that inhibiting vacuolar-ATPase using the macrolide bafilomycin A1 was the only agent able to potently block viral entry without associated cellular toxicity. Using both pseudotyped and authentic virus, we showed that bafilomycin A1 inhibits SARS-CoV-2 infection both in the absence and presence of TMPRSS2. Moreover, synergy was observed upon combining bafilomycin A1 with Camostat, a TMPRSS2 inhibitor, in neutralizing SARS-CoV-2 entry into TMPRSS2-expressing cells. Overall, this study highlights the importance of the endosomal route of entry for SARS-CoV-2 and provides a rationale for the generation of successful intervention strategies against this virus that combine inhibitors of both entry pathways.

Longitudinal analysis of SARS-CoV-2 reinfection reveals distinct kinetics and emergence of cross-neutralizing antibodies to variants of concern.

The ongoing evolution of SARS-CoV-2 continues to raise new questions regarding the duration of immunity to reinfection with emerging variants. To address these knowledge gaps, controlled investigations in established animal models are needed to assess duration of immunity induced by each SARS-CoV-2 lineage and precisely evaluate the extent of cross-reactivity and cross-protection afforded. Using the Syrian hamster model, we specifically investigated duration of infection acquired immunity to SARS-CoV-2 ancestral Wuhan strain over 12 months. Plasma spike- and RBD-specific IgG titers against ancestral SARS-CoV-2 peaked at 4 months post-infection and showed a modest decline by 12 months. Similar kinetics were observed with plasma virus neutralizing antibody titers which peaked at 2 months post-infection and showed a modest decline by 12 months. Reinfection with ancestral SARS-CoV-2 at regular intervals demonstrated that prior infection provides long-lasting immunity as hamsters were protected against severe disease when rechallenged at 2, 4, 6, and 12 months after primary infection, and this coincided with the induction of high virus neutralizing antibody titers. Cross-neutralizing antibody titers against the B.1.617.2 variant (Delta) progressively waned in blood over 12 months, however, re-infection boosted these titers to levels equivalent to ancestral SARS-CoV-2. Conversely, cross-neutralizing antibodies to the BA.1 variant (Omicron) were virtually undetectable at all time-points after primary infection and were only detected following reinfection at 6 and 12 months. Collectively, these data demonstrate that infection with ancestral SARS-CoV-2 strains generates antibody responses that continue to evolve long after resolution of infection with distinct kinetics and emergence of cross-reactive and cross-neutralizing antibodies to Delta and Omicron variants and their specific spike antigens.

Analysis of mutations in multiple loci of Neisseria gonorrhoeae isolates reveals effects of PIB, PBP2 and MtrR on reduced susceptibility to ceftriaxone.

OBJECTIVES: To elucidate loci in Neisseria gonorrhoeae implicated in reduced susceptibility to ceftriaxone. METHODS: N. gonorrhoeae isolates were collected in Shanghai, China, in 2005 and 2008. Twenty-eight isolates with reduced susceptibility to ceftriaxone (CRO(Red); MIC = 0.125-0.25 mg/L) were studied for mutations in PorB (porB), MtrR (mtrR), PBP2 (penA) and PBP1 (ponA). The mutation profiles of the 28 CRO(Red) isolates were compared with those of 32 ceftriaxone-susceptible isolates (CRO(S); MIC = 0.004-0.016 mg/L). porB-based DNA sequence typing and N. gonorrhoeae multi-antigen sequence typing (NG-MAST) analyses were performed. RESULTS: Significantly more CRO(Red) isolates (89.3%) exhibited a PIB phenotype as compared with the CRO(S) isolates (59.4%) (P = 0.02). Double mutations (G45D/H105Y or A39T/H105Y) in MtrR were associated with CRO(Red) phenotypes. A 'wild-type' MtrR protein characterized CRO(Red) isolates (50.0%, 14/28), while a single H105Y mutation was observed only in CRO(S) isolates (43.8%, 14/32). Both CRO(Red) and CRO(S) isolates carried an '-A' deletion in the mtrR promoter. Six of 15 mutation patterns observed in PBP2 were new. Mutation patterns XIII (17.9% of CRO(Red) isolates) and XVII or XVIII (25.0% of CRO(Red) isolates) of PBP2 comprised A501V/G542S or A501V/P551S double mutations and were associated with a CRO(Red) phenotype. The mosaic PBP2 (pattern X) was not observed. The L421P mutation in PBP1 was observed in all CRO(Red) and in 97.0% of CRO(S) isolates. CRO(Red) isolates were non-clonal. CONCLUSIONS: Reduced susceptibility to ceftriaxone in N. gonorrhoeae is mediated by porB1b alleles and is associated with specific mutations in PBP2 and in the DNA binding and dimerization domains of MtrR.

A minD mutant of enterohemorrhagic E. coli O157:H7 has reduced adherence to human epithelial cells.

Adherence to epithelial cells is a prerequisite for intestinal colonization by the bacterial pathogen, enterohemorrhagic Escherichia coli (EHEC). The deletion of minD, a cell division gene, in EHEC caused reduced adherence to human epithelioid cervical carcinoma (HeLa) and human colonic adenocarcinoma (Caco-2) cells as compared to wild-type. The minD mutant formed minicells and filaments owing to aberrant cytokinesis. Moreover, its ability to form microcolonies as typically seen in the co-cultures of wild-type with Caco-2 cells, was abolished. In conclusion, the present study highlights the importance of minD in regards to EHEC adherence to human epithelial cells.

Antimicrobial activity of flavonoids from Piper lanceaefolium and other Colombian medicinal plants against antibiotic susceptible and resistant strains of Neisseria gonorrhoeae.

BACKGROUND: The successful treatment of Neisseria gonorrhoeae (NG) infections is increasingly problematic because of the resistance of this pathogen to multiple antimicrobial agents. This development underscores the need for new antimicrobial sources. In the current study, 21 crude methanol extracts, from 19 plants used in Colombian traditional medicine for cutaneous infections, were screened for antimicrobial activity against NG. METHODS: Extracts were screened by disc susceptibility assay. In addition, the minimum inhibitory concentrations of active compounds from P. lanceaefolium were assayed using a panel of 26 NG strains comprising 12 antibiotic-resistant phenotypes. RESULTS: In all, 71% of the crude extracts exhibited antibacterial activity against the antibiotic susceptible NG strain WHO V, whereas 10% of the extracts inhibited penicillinase-producing NG strain GC1-182. The crude extract of Piper lanceaefolium was the only extract to show significant activity without ultraviolet (UV) light activation. Preliminary screening identified 3 compounds in this plant possessing antimicrobial activity: the flavonoids 5,7-dihydroxyflavanone (pinocembrin), 2',4',6'-trihydroxychalcone (pinocembrin chalcone), and the prenylated benzoic acid derivative cyclolanceaefolic acid methyl ester. Pinocembrin and pinocembrin chalcone inhibited 100% of the NG panel at 64 µg/mL and 128 µg/mL, respectively, whereas cyclolanceaefolic acid methyl ester inhibited 44% of the strains at 128 µg/mL. CONCLUSIONS: This is the first report of the antibacterial activity of Columbian plants against NG. The activity of the 2 flavonoids, pinocembrin, and pinocembrin chalcone, toward both susceptible and resistant NG strains makes them promising candidates for further research.

An enveloped virus-like particle vaccine expressing a stabilized prefusion form of the SARS-CoV-2 spike protein elicits highly potent immunity.

We evaluated enveloped virus-like particles (eVLPs) expressing various forms of the Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein and several adjuvants in an effort to identify a highly potent Coronavirus disease 2019 (COVID-19) vaccine candidate. eVLPs expressing a modified prefusion form of SARS-CoV-2 spike protein were selected as they induced high antibody binding titers and neutralizing activity after a single injection in mice. Formulation of SARS-CoV-2 S eVLPs with aluminum phosphate resulted in balanced induction of IgG2 and IgG1 isotypes and antibody binding and neutralization titers were undiminished for more than 3 months after a single immunization. A single dose of this candidate, named VBI-2902a, protected Syrian golden hamsters from challenge with SARS-CoV-2 and supports the on-going clinical evaluation of VBI-2902a as a highly potent vaccine against COVID-19.

EF1025, a Hypothetical Protein From Enterococcus faecalis, Interacts With DivIVA and Affects Cell Length and Cell Shape.

DivIVA plays multifaceted roles in Gram-positive organisms through its association with various cell division and non-cell division proteins. We report a novel DivIVA interacting protein in Enterococcus faecalis, named EF1025 (encoded by EF1025), which is conserved in Gram-positive bacteria. The interaction of EF1025 with DivIVAEf was confirmed by Bacterial Two-Hybrid, Glutathione S-Transferase pull-down, and co-immunoprecipitation assays. EF1025, which contains a DNA binding domain and two Cystathionine ß-Synthase (CBS) domains, forms a decamer mediated by the two CBS domains. Viable cells were recovered after insertional inactivation or deletion of EF1025 only through complementation of EF1025 in trans. These cells were longer than the average length of E. faecalis cells and had distorted shapes. Overexpression of EF1025 also resulted in cell elongation. Immuno-staining revealed comparable localization patterns of EF1025 and DivIVAEf in the later stages of division in E. faecalis cells. In summary, EF1025 is a novel DivIVA interacting protein influencing cell length and morphology in E. faecalis.

Comparison of Neisseria gonorrhoeae multiantigen sequence typing and porB sequence analysis for identification of clusters of N. gonorrhoeae isolates.

porB DNA sequence analysis and Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST) methods were compared for their abilities to discriminate strains and to identify epidemiologically congruent pairs of N. gonorrhoeae. Both methods provided high-level discrimination of strains. NG-MAST further differentiated large porB-based clusters. However, considerations of cost suggest that porB DNA sequence analysis is a useful tool for preliminary molecular analysis of the epidemiology of N. gonorrhoeae.

Clusters of circulating Neisseria gonorrhoeae strains and association with antimicrobial resistance in Shanghai.

OBJECTIVES: (i) To distinguish Neisseria gonorrhoeae isolates in Shanghai by porB typing; (ii) to ascertain the congruence of porB DNA sequence typing with cases linked epidemiologically; (iii) to determine the association of specific PorB mutations with antimicrobial resistance to penicillin or tetracycline. METHODS: porB DNA sequences of 174 N. gonorrhoeae isolates, collected from 143 male patients and 31 female sexual partners in Shanghai were determined. Phylogenetic analysis was used to determine sequence associations and concordance with epidemiologically linked cases. PorB protein sequences were compared with the wild-type sequence to identify mutations associated with antimicrobial resistance to penicillin and tetracycline. RESULTS: porB1a genotypes comprised 27.0% of the isolates and included 15 distinct DNA sequences, while 73.0% of the isolates carried porB1b genotypes with 63 distinct DNA sequences. porB DNA sequence typing was congruent with patient-reported sexual contacts. In addition, porB DNA sequence analysis revealed a number of strains with identical DNA sequences not identified through traditional epidemiological methods. The porB1b isolates had a significantly higher percentage of chromosomally mediated resistance to tetracycline and higher MIC50s to penicillin and ciprofloxacin. G120K/A121D mutations were observed in 71.1% of PIB isolates and were associated with resistance to penicillin and/or tetracycline. The majority of the PIA isolates (82.1%) also carried G120D/A121G double mutations. The index of discrimination for porB DNA sequence analysis was 95%. CONCLUSIONS: The porB1b genotype was found to be predominant in Shanghai. porB DNA sequence typing was sufficiently discriminatory for differentiating N. gonorrhoeae isolates and was congruent with epidemiological linkages. Novel porB sequences of N. gonorrhoeae and novel mutations of PorB proteins were identified.

Identification of the coiled-coil domains of Enterococcus faecalis DivIVA that mediate oligomerization and their importance for biological function.

Bacillus subtilis (Bs) DivIVA comprises coiled-coil structures and self-associates forming a 10-12 mer complex in vitro. Using bioinformatic approaches, we determined that Enterococcus faecalis (Ef) DivIVA comprises four coiled-coil domains, one at the N-terminus, the second and the third in the central region of the protein and the fourth at the C-terminus. We determined that DivIVA(Ef) self-interacts and forms a 10-12 multimeric complex. Point mutations or deletions of the central regions predicted bioinformatically to disrupt the coiled-coil structures either eliminated or weakened DivIVA(Ef) self-interaction and reduced oligomerization. Mutations disrupting the N- and C-terminal coiled-coils of DivIVA(Ef) did not affect DivIVA(Ef) oligomerization. The introduction of DivIVA(Ef) mutations to both the N-terminal and the central coiled-coil domains were lethal unless rescued by expressing wild-type DivIVA(Ef) in trans. E. faecalis cells expressing these mutations displayed aberrant cell morphology, indicating disruption of the normal cell division phenotype. The results in E. faecalis also indicate that both the N-terminal and the central coiled-coil structures of DivIVA(Ef) are indispensable for proper biological function. Overexpression of wild-type DivIVA(Ef) in both rod-shaped and round Escherichia coli cells resulted in morphological changes, while the overexpression of DivIVA(Ef) mutations failed to induce such alterations.

Behavioral and socioeconomic risk factors associated with probable resistance to ceftriaxone and resistance to penicillin and tetracycline in Neisseria gonorrhoeae in Shanghai.

Globally, incidence of Neisseria gonorrhoeae infection is once again the highest of the bacterial sexually transmitted infections. The bacterium can produce serious complications in those infected, and emerging resistance to third generation cephalosporins could usher in an era of potentially untreatable gonorrhea. This research aimed to identify risk factors for antibiotic resistant gonorrhea infection among clients at a Shanghai sexually transmitted infection clinic over two time periods, 2004-2005 and 2008-2011. Demographic and risk factor behavior data, and biological samples for antimicrobial resistance analysis, were collected. Statistical models were built to identify risk factors associated with probable resistance to ceftriaxone and resistance to penicillin and tetracycline. High levels of ciprofloxacin resistance (98%) in our sample precluded examining its risk factors; all isolates were susceptible to spectinomycin. Overall (P<0.001), chromosomal (P<0.001), and plasmid-mediated (P = 0.01) penicillin resistance decreased from the first to second period of the study. For tetracycline, chromosomal resistance decreased (P = 0.01) and plasmid-mediated resistance increased (P<0.001) between the first and second periods of study. In multi-level multivariable regression models, male gender (P = 0.03) and older age (P = 0.01) were associated with increased minimum inhibitory concentrations to ceftriaxone. Male gender (P = 0.03) and alcohol use (P = 0.02) were associated with increased odds of overall tetracycline resistance. Male gender was associated with increased odds of chromosomally-mediated tetracycline resistance (P = 0.04), and alcohol use was associated with increased odds of plasmid-mediated tetracycline resistance (P = 0.02). Additionally, individuals in middle-salary categories were found to have lower odds of plasmid-mediated resistance to tetracycline compared with those in the lowest salary category (P≤0.02). This study is one of the first to use multilevel analysis to consider the association between risk factors for gonorrhea infections and mechanisms of resistance to individual antibiotics. Such information is urgently needed to combat the growing threat of untreatable gonorrhea.

Regulation of minD by oxyR in Neisseria gonorrhoeae.

In Neisseria gonorrhoeae, cytokinesis involves Escherichia coli homologues of minC, minD and minE which are encoded as part of a min operon. MinD, a 30 kD protein component of the MinC-MinD septum inhibitory complex, together with MinE, mediates cell division site selection. Gonococci mutated in minD display aberrant cytokinesis, abnormal morphology, defective microcolony formation and virulence. minD is 274 bp upstream of oxyR, another min operon gene in N. gonorrhoeae, which encodes a redox-responsive transcriptional regulator implicated in responses to oxidative stress. In this study, we aimed to examine the oxyR-mediated regulation of minD. We observed the cotranscription of oxyR with the minCDE gene cluster. The mutation of oxyR resulted in non-midline formation of the division septum, anomalous DNA segregation, and increased aggregation of bacterial cells. qRT-PCR and Western Blot analysis revealed upregulation of minD in an oxyR mutant as compared to its isogenic wild-type N. gonorrhoeae strain in stationary phase. Furthermore, the exposure to oxidative stress in the form of H2O2 increased MinD expression levels in wild-type N. gonorrhoeae. Using ß-galactosidase activity-based promoter assays, we found that oxyR negatively regulates the promoter region (PminD) upstream of minD. Our results demonstrate the involvement of oxyR in cell division and minD expression in N. gonorrhoeae.

Influence of conserved and hypervariable genetic markers on genotyping circulating strains of Neisseria gonorrhoeae.

Presently there is no vaccine against Neisseria gonorrhoeae and therefore accurate information on gonococcal transmission plays a crucial role for interventions designed to limit the spread of infections caused by this microorganism. We evaluated the impact of two different categories of genetic markers, (i) concatenated sequences of 10 housekeeping genes and (ii) hypervariable porB DNA sequences, on the genetic relatedness and subsequently on genotyping analysis of this human pathogen. Eighty gonococcal isolates from Canada, China, the US, Argentina, Venezuela and Chile, collected over different times, were analyzed. Our results show that the choice of genetic marker had a profound effect on the interpretation of genotyping results associated with N. gonorrhoeae. The concatenated sequences of the housekeeping genes preserved the genetic relatedness of closely related isolates, enabling detection of the predominant strains circulating within a community (Saskatchewan, Canada) over an extended period of time. In contrast, a genetic marker based on antigen gene, porB, may lead to a failure to detect these predominant circulating strains. Based on the analysis of the DNA sequences of the 10 housekeeping genes, we identified two major clonal complexes, CC33 and CC22, which comprised STs from China, and Argentina as well as two STs from Canada. Several minor clonal complexes were observed among isolates from Saskatchewan. eBURST analysis suggested that the majority of the tested gonococcal isolates from Saskatchewan, Canada were endemic, with only a couple of genotypes introduced.

Antimicrobial susceptibility and molecular determinants of quinolone resistance in Neisseria gonorrhoeae isolates from Shanghai.

OBJECTIVES: To determine the antimicrobial susceptibility of Neisseria gonorrhoeae from Shanghai and to type the quinolone resistance-determining regions (QRDRs) of ciprofloxacin-resistant isolates. METHODS: N. gonorrhoeae isolates (n = 159) were consecutively collected from male patients in Shanghai and examined for their antimicrobial susceptibilities to penicillin, tetracycline, ciprofloxacin, spectinomycin and ceftriaxone. The mutation profiles of the QRDRs of gyrA and parC were determined for 103 isolates including one susceptible isolate and one isolate with intermediate levels of susceptibility to ciprofloxacin. RESULTS: High percentages of the 159 isolates were resistant to ciprofloxacin (98.7%), penicillin (93.1%) and tetracycline (56.5%). Penicillinase-producing N. gonorrhoeae (PPNG, 37.8%) or penicillinase-producing/tetracycline-resistant N. gonorrhoeae (PP/TRNG, 13.8%) accounted for 51.6% of the isolates. Chromosomal resistance to penicillin was observed in 41.5% of the isolates. Tetracycline resistance was noted in 56.5% of the isolates with 20.1% carrying plasmid-mediated resistance and 36.4% being chromosomally resistant. All isolates were susceptible to ceftriaxone and spectinomycin, although a trend to decreased susceptibility was noted. QRDR mutations were observed in the 101 ciprofloxacin-resistant isolates and the one ciprofloxacin-intermediate isolate, in contrast to the ciprofloxacin-susceptible isolate tested. Mutations in the QRDRs comprised four predominant (65.0% of the 103 isolates) patterns of a total of 19 patterns. Mutations in parC were significantly associated with higher MICs of ciprofloxacin. CONCLUSIONS: Spectinomycin and ceftriaxone are currently recommended for the treatment of gonorrhoea in Shanghai. Although the present study indicates that these antimicrobials should remain effective, the identification of isolates with decreased susceptibility underscores the importance of ongoing antimicrobial susceptibility surveillance to monitor and respond to the emergence of resistant isolates.

Enterococcus faecalis divIVA: an essential gene involved in cell division, cell growth and chromosome segregation.

Enterococcus faecalis divIVA (divIVAEf) is an essential gene implicated in cell division and chromosome segregation. This gene was disrupted by insertional inactivation creating E. faecalis JHSR1, which was viable only when a wild-type copy of divIVAEf was expressed in trans, confirming the essentiality of the gene. The absence of DivIVAEf in E. faecalis JHSR1 inhibited proper cell division, which resulted in abnormal cell clusters possessing enlarged cells of altered shape instead of the characteristic diplococcal morphology of enterococci. The lower viability of the divIVAEf mutant is caused by improper nucleoid segregation and impaired septation within the numerous cells generated in each cluster. Overexpression of DivIVAEf in Escherichia coli KJB24 resulted in enlarged cells with disrupted cell division, suggesting that this round E. coli mutant strain could be used as an indicator for functionality of DivIVAEf. A Bacillus subtilis divIVA mutant was not complemented by DivIVAEf, indicating that this protein does not recognize DivIVA-specific target sites in B. subtilis, or that it does not interact with other proteins of the cell division machinery of this micro-organism. DivIVAEf also failed to complement a Streptococcus pneumoniae divIVA mutant, supporting the phylogenetic distance between Enterococcus and Streptococcus. Our results indicate that DivIVA is a species-specific multifunctional protein implicated in cell division and chromosome segregation in E. faecalis.

Garlic natural health products exhibit variable constituent levels and antimicrobial activity against Neisseria gonorrhoeae, Staphylococcus aureus and Enterococcus faecalis.

The composition of 19 garlic natural health products (NHPs) and fresh garlic extracts were determined, as was their antibacterial activity. The 19 NHPs and 5 fresh garlic extract standards were analysed for their principal active constituents. They were also extracted for 5, 10 or 15 min in water to fresh garlic equivalents of 200 mg/mL. The extract's minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) against three indicator microorganisms (Neisseria gonorrhoeae, Staphylococcus aureus and Enterococcus faecalis) were determined by the broth microdilution method. While 47% of the aqueous garlic NHP extracts exhibited activity against N. gonorrhoeae, only 16% of the aqueous extracts inhibited S. aureus or E. faecalis at all three timepoints. Generally, products with high antimicrobial activity contained higher levels of garlic constituents with comparable activity to fresh garlic extracts, while products with marginal antibacterial activity often contained lower concentrations of constituents than their product labels indicated. Different extraction times affected antibacterial activity only against N. gonorrhoeae and tended to be correlated with levels of allicin. Thus, many extracts showed discrepancies in both composition, allicin:alliin ratio and antimicrobial activity, raising concerns as to standards of preparation and quality control for these products.