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ABSTRACT The minimum inhibitory concentration (MIC) of echinocandins against Aspergillus spp. does not represent the actual inhibition threshold of echinocandins. Therefore, the recommended method to evaluate their activity is determining the minimum effective concentration (MEC) in broth microdilution, a method that is less common in clinical settings. This study aimed to assess a user-friendly commercial method, Sensititre YeastOne (SYO), to determine the effectiveness of echinocandins (caspofungin, anidulafungin and micafungin) against Aspergillus spp. Echinocandins MEC was determined against 23 isolates of Aspergillus spp. using SYO and the reference Clinical and Laboratory Standards Institute (CLSI) method. MECs were read with an inverted microscope and a reading mirror. Essential agreement (EA) between the tested methods was defined as a ±twofold dilution difference. There was a high EA (91%–100%) between the reference method and SYO in determining echinocandins MEC against Aspergillus isolates using inverted microscopy. A high EA was also observed between SYO MEC determined by inverted microscopy and a reading mirror, but different incubation times were required. SYO is a reliable, simple method for determining the MEC of echinocandins against Aspergillus isolates, preferably with an inverted microscope, and can be easily used in clinical laboratories when echinocandin susceptibility testing is required.IMPORTANCEUsing a commercial method such as Sensititre YeastOne (SYO) to determine the minimum effective concentration (MEC) of echinocandins against Aspergillus spp. has been shown to be a reliable alternative to the Clinical and Laboratory Standards Institute (CLSI) reference method. This makes it more suitable for high-volume clinical laboratories. SYO provides accurate results comparable to the standard method and could potentially improve patient care by guiding more optimal antifungal treatment choices for patients with Aspergillus infections.

Enteropathogenic E. coli (EPEC) is a Gram-negative bacterial pathogen that causes persistent diarrhea. Upon attachment to the apical plasma membrane of the intestinal epithelium, the pathogen translocates virulence proteins called effectors into the infected cells. These effectors hijack numerous host processes for the pathogen’s benefit. Therefore, studying the mechanisms underlying their action is crucial for a better understanding of the disease. We show that translocated EspH interacts with multiple host Rab GTPases. AlphaFold predictions and site-directed mutagenesis identified glutamic acid and lysine at positions 37 and 41 as Rab interacting residues in EspH. Mutating these sites abolished the ability of EspH to inhibit Akt and mTORC1 signaling, lysosomal exocytosis, and bacterial invasion. Knocking out the endogenous Rab8a gene expression highlighted the involvement of Rab8a in Akt/mTORC1 signaling and lysosomal exocytosis. A phosphoinositide binding domain with a critical tyrosine was identified in EspH. Mutating the tyrosine abolished the localization of EspH at infection sites and its capacity to interact with the Rabs. Our data suggest novel EspH-dependent mechanisms that elicit immune signaling and membrane trafficking during EPEC infection.

Abstract The humoral response after the fourth dose of a mRNA vaccine against COVID-19 has not been adequately described in elderly recipients, particularly those not exposed previously to SARS-CoV-2. Serum anti-RBD IgG levels (Abbott SARS-CoV-2 IgG II Quant assay) and neutralizing capacities (spike SARS-CoV-2 pseudovirus Wuhan and Omicron BA.1 variant) were measured after the third and fourth doses of a COVID-19 mRNA vaccine among 46 elderly residents (median age 85 years [IQR 81; 89]) of an assisted living facility. Among participants never infected by SARS-CoV-2, the mean serum IgG levels against RBD (2025 BAU/ml), 99 days after the fourth vaccine, was as high as 76 days after the third vaccine (1987 BAU/ml), and significantly higher (p = 0.030) when the latter were corrected for elapsed time. Neutralizing antibody levels against the historical Wuhan strain were significantly higher (Mean 1046 vs 1573; p = 0.002) and broader (against Omicron) (Mean 170 vs 375; p = 0.018), following the fourth vaccine. The six individuals with an Omicron breakthrough infection mounted strong immune responses for anti-RBD and neutralizing antibodies against the Omicron variant indicating that the fourth vaccine dose did not prevent a specific adaptation of the immune response. These findings point out the value of continued vaccine boosting in the elderly population

The mechanism of flavivirus activation for membrane fusion is not yet understood. Here, Vaney et al. describe how the viral pr protein, derived from an HSP40/DnaJ chaperonin, interacts with a pH sensing loop in the envelope protein E for fusogenic activation.

Intramuscularly administered vaccines stimulate robust serum neutralizing antibodies, yet they are often less competent in eliciting sustainable “sterilizing immunity” at the mucosal level. Our study uncovers a strong temporary neutralizing mucosal component of immunity, emanating from intramuscular administration of an mRNA vaccine. We show that saliva of BNT162b2 vaccinees contains temporary IgA targeting the receptor-binding domain (RBD) of severe acute respiratory syndrome coronavirus-2 spike protein and demonstrate that these IgAs mediate neutralization. RBD-targeting IgAs were found to associate with the secretory component, indicating their bona fide transcytotic origin and their polymeric multivalent nature. The mechanistic understanding of the high neutralizing activity provided by mucosal IgA, acting at the first line of defense, will advance vaccination design and surveillance principles and may point to novel treatment approaches and new routes of vaccine administration and boosting.

Dengue is a rapidly emerging, mosquito-borne viral infection, with an estimated 400 million infections occurring annually. To gain insight into dengue immunity, we characterized 145 human monoclonal antibodies (mAbs) and identified a previously unknown epitope, the envelope dimer epitope (EDE), that bridges two envelope protein subunits that make up the 90 repeating dimers on the mature virion. The mAbs to EDE were broadly reactive across the dengue serocomplex and fully neutralized virus produced in either insect cells or primary human cells, with 50% neutralization in the low picomolar range. Our results provide a path to a subunit vaccine against dengue virus and have implications for the design and monitoring of future vaccine trials in which the induction of antibody to the EDE should be prioritized.

The immunodominant epitope of dengue virus envelope protein (E) induces poorly neutralizing antibodies, which poses a problem for vaccine development. Here, the authors engineer covalently locked E dimers exposing an epitope that has been shown to induce potent and broadly neutralizing antibodies.

When grown on a solid surface, bacteria form highly organized colonies, yet little is known about the earliest stages of colony establishment. Following Bacillus subtilis colony development from a single progenitor cell, a sequence of highly ordered spatiotemporal events was revealed. Colony was initiated by the formation of leading-cell chains, deriving from the colony center and extending in multiple directions, typically in a “Y-shaped” structure. By eradicating particular cells during these early stages, we could influence the shape of the resulting colony and demonstrate that Y-arm extension defines colony size. A mutant in ymdB encoding a phosphodiesterase displayed unordered developmental patterns, indicating a role in guiding these initial events. Finally, we provide evidence that intercellular nanotubes contribute to proper colony formation. In summary, we reveal a “construction plan” for building a colony and provide the initial molecular basis for this process.

Many viruses transmitted by blood-sucking arthropods have emerged worldwide and cause serious human and animal diseases. Arthropod-borne (arbo-)viruses possess a peculiar capacity to replicate in very different hosts, such as mosquitoes and mammals (e.g., dengue and chikungunya). However, the molecular mechanisms that enable arboviruses to enter and replicate in very different hosts—such as arthropods, mammals, and birds—remain highly enigmatic. Insect-restricted viruses provide a reference point regarding this issue, since they lack the strategies of infecting vertebrate hosts and are viewed as evolutionary precursors of arboviruses. Negev viruses comprise a recently discovered insect-restricted taxon exhibiting worldwide distribution among blood sucking arthropods. Negev viruses are enveloped, positive sense, nonsegmented RNA viruses. The virus genome is ca. 10 kb long and contains three ORFs, with the longest ORF1 (7 kb) encoding putative replicative enzymes. ORF2 (1.2 kb) and ORF3 (0.7 kb) do not have any clear homologs and are predicted to encode membrane proteins. We explored the functional and structural aspects of Negev viruses and the role of their membrane glycoproteins during Negev virus entry into the mosquito cells. We employ biochemistry, structural biology, and microscopy to reveal mechanisms of Negev virion organization, and its entry into host cells followed by fusion with the host membranes to allow subsequent genome delivery and replication.

Negeviruses (NVs) are a recently discovered taxon of enveloped, positive sense, single-stranded RNA viruses, infecting blood-sucking insects. While classical arthropod-borne (arbo)viruses like dengue and Chikungunya infect both insects and vertebrates, NVs are restricted to insects and do not have any known vertebrate host and are thus classified as insect-restricted viruses. Previous works have predicted a structure consisting of three ORFs, the first with homologous regions to RNA-dependent RNA polymerase, helicase, and methyl transferases in plant viruses. On the contrary, ORF2 and ORF3 do not have homologs and are predicted to encode membrane glycoproteins. Their structures, functions, and significance remain vague. We focus on the characterization of the viral proteins, structural organization of the virion, and the principles of their interaction with the host cell. We purified the virion particles of Negev virus produced in mosquito cells and identified its structural components. In addition, we cloned and overexpressed ORF2 and ORF3 of Negeviruses. Furthermore, we defined and successfully produced and purified recombinant ORF2. Subsequent characterization using gel filtration, ion exchange, and MALS techniques revealed that the ORF2 of Negeviruses exhibit different higher order assembly patterns: dimerization and multimerization in a concentration- and pH-dependent manner that correspond to their biological role. We combine biochemical, structural and cell biology techniques to unravel mechanisms of Negev virus interaction with the host cell.

ABSTRACT Direct interactions between bacterial and host glycans have been recently reported to be involved in the binding of pathogenic bacteria to host cells. In the case of Shigella, the Gram-negative enteroinvasive bacterium responsible for acute rectocolitis, such interactions contribute to bacterial adherence to epithelial cells. However, the role of glycans in the tropism of Shigella for immune cells whose glycosylation pattern varies depending on their activation state is unknown. We previously reported that Shigella targets activated, but not nonactivated, human CD4+ T lymphocytes. Here, we show that nonactivated CD4+ T lymphocytes can be turned into Shigella-targetable cells upon loading of their plasma membrane with sialylated glycosphingolipids (also termed gangliosides). The Shigella targeting profile of ganglioside-loaded nonactivated T cells is similar to that of activated T cells, with a predominance of injection of effectors from the type III secretion system (T3SS) not resulting in cell invasion. We demonstrate that gangliosides interact with the O-antigen polysaccharide moiety of lipopolysaccharide (LPS), the major bacterial surface antigen, thus promoting Shigella binding to CD4+ T cells. This binding step is critical for the subsequent injection of T3SS effectors, a step which we univocally demonstrate to be dependent on actin polymerization. Altogether, these findings highlight the critical role of glycan-glycan interactions in Shigella pathogenesis. IMPORTANCE Glycosylation of host cell surface varies with species and location in the body, thus contributing to species specificity and tropism of microorganisms. Cross talk by Shigella, the Gram-negative enteroinvasive bacterium responsible for bacillary dysentery, with its exclusively human host has been extensively studied. However, the molecular determinants of the step of binding to host cells are poorly defined. Taking advantage of the observation that human-activated CD4+ T lymphocytes, but not nonactivated cells, are targets of Shigella, we succeeded in rendering the refractory cells susceptible to targeting upon loading of their plasma membrane with sialylated glycosphingolipids (gangliosides) that are abundantly present on activated cells. We show that interactions between the sugar polar part of gangliosides and the polysaccharide moiety of Shigella lipopolysaccharide (LPS) promote bacterial binding, which results in the injection of effectors via the type III secretion system. Whereas LPS interaction with gangliosides was proposed long ago and recently extended to a large variety of glycans, our findings reveal that such glycan-glycan interactions are critical for Shigella pathogenesis by driving selective interactions with host cells, including immune cells.

Zika virus is a member of the Flavivirus genus that had not been associated with severe disease in humans until the recent outbreaks, when it was linked to microcephaly in newborns in Brazil and to Guillain-Barre syndrome in adults in French Polynesia. Zika virus is related to dengue virus, and here we report that a subset of antibodies targeting a conformational epitope isolated from patients with dengue virus also potently neutralize Zika virus. The crystal structure of two of these antibodies in complex with the envelope protein of Zika virus reveals the details of a conserved epitope, which is also the site of interaction of the envelope protein dimer with the precursor membrane (prM) protein during virus maturation. Comparison of the Zika and dengue virus immunocomplexes provides a lead for rational, epitope-focused design of a universal vaccine capable of eliciting potent cross-neutralizing antibodies to protect simultaneously against both Zika and dengue virus infections., Zika virus (ZIKV) is an arthropod-borne enveloped virus belonging to the Flavivirus genus in the family Flaviviridae, which also includes the human pathogenic yellow fever, dengue, West Nile and tick-borne [...]