Beschreibung: The morbillivirus cell entry machinery consists of a fusion (F) protein trimer that refolds to mediate membrane fusion following receptor-induced conformational changes in its binding partner, the tetrameric attachment (H) protein. To identify molecular determinants that control F refolding, we generated F chimeras between measles virus (MeV) and canine distemper virus (CDV). We located a central pocket in the globular head domain of CDV F that regulates the stability of the metastable, prefusion conformational state of the F trimer. Most mutations introduced into this "pocket'" appeared to mediate a destabilizing effect, a phenotype associated with enhanced membrane fusion activity. Strikingly, under specific triggering conditions (i.e., variation of receptor type and H protein origin), some F mutants also exhibited resistance to a potent morbillivirus entry inhibitor, which is known to block F triggering by enhancing the stability of prefusion F trimers. Our data reveal that the molecular nature of the F stimulus and the intrinsic stability of metastable prefusion F both regulate the efficiency of F refolding and escape from small-molecule refolding blockers. IMPORTANCE With the aim to better characterize the thermodynamic basis of morbillivirus membrane fusion for cell entry and spread, we report here that the activation energy barrier of prefusion F trimers together with the molecular nature of the triggering "stimulus" (attachment protein and receptor types) define a "triggering range," which governs the initiation of the membrane fusion process. A central "pocket" microdomain in the globular F head contributes substantially to the regulation of the conformational stability of the prefusion complexes. The triggering range also defines the mechanism of viral escape from entry inhibitors and describes how the cellular environment can affect membrane fusion efficiency.

Beschreibung: Schistosomiasis is a chronic and debilitating disease caused by a trematode of the genus Schistosoma and affects over 207 million people. Chemotherapy is the only immediate recourse for minimizing the prevalence of this disease and involves predominately the administration of a single drug, praziquantel (PZQ). Although PZQ has proven efficacy, there is a recognized need to develop new drugs as schistosomicides since studies have shown that repeated use of this drug in areas of endemicity may cause a temporary reduction in susceptibility in isolates of Schistosoma mansoni . Hydrazones, thiosemicarbazones, phthalimides, and thiazoles are thus regarded as privileged structures used for a broad spectrum of activities and are potential candidates for sources of new drug prototypes. The present study determined the in vitro schistosomicidal activity of 10 molecules containing these structures. During the assays, parameters such motility and mortality, oviposition, morphological changes in the tegument, cytotoxicity, and immunomodulatory activity caused by these compounds were evaluated. The results showed that compounds formed of thiazole and phthalimide led to higher mortality of worms, with a significant decline in motility, inhibition of pairing and oviposition, and a mortality rate of 100% starting from 144 h of exposure. These compounds also stimulated the production of nitric oxide and tumor necrosis factor alpha (TNF-α), thereby demonstrating the presence of immunomodulatory activity. The phthalyl thiazole LpQM-45 caused significant ultrastructural alterations, with destruction of the tegument in both male and female worms. According to the present study, phthalyl thiazole compounds possess antischistosomal activities and should form the basis for future experimental and clinical trials.

Beschreibung: Membrane fusion for morbillivirus cell entry relies on critical interactions between the viral fusion (F) and attachment (H) envelope glycoproteins. Through extensive mutagenesis of an F cavity recently proposed to contribute to F's interaction with the H protein, we identified two neighboring hydrophobic residues responsible for severe F-to-H binding and fusion-triggering deficiencies when they were mutated in combination. Since both residues reside on one side of the F cavity, the data suggest that H binds the F globular head domain sideways.

Beschreibung: Streptococcus sanguinis is a pioneer species of teeth and a common opportunistic pathogen of infective endocarditis. In this study, we identified a two-component system, S. sanguinis SptRS (SptRS Ss ), affecting S. sanguinis survival in saliva and biofilm formation. Isogenic mutants of sptR Ss (SKsptR) and sptS Ss (SKsptS) showed reduced cell counts in ex vivo assays of viability in saliva compared to those of parent strain SK36 and complemented mutants. Reduced counts of the mutants in saliva were associated with reduced growth rates in nutrient-poor medium (RPMI) and increased susceptibility to the deposition of C3b and the membrane attach complex (MAC) of the complement system, a defense component of saliva and serum. Conversely, sptR Ss and sptS Ss mutants showed increased biofilm formation associated with higher levels of production of H 2 O 2 and extracellular DNA. Reverse transcription-quantitative PCR (RT-qPCR) comparisons of strains indicated a global role of SptRS Ss in repressing genes for H 2 O 2 production (2.5- to 15-fold upregulation of spxB , spxR , vicR , tpk , and ackA in sptR Ss and sptS Ss mutants), biofilm formation, and/or evasion of host immunity (2.1- to 11.4-fold upregulation of srtA , pcsB , cwdP , iga , and nt5e ). Compatible with the homology of SptR Ss with AraC-type regulators, duplicate to multiple conserved repeats were identified in 1,000-bp regulatory regions of downstream genes, suggesting that SptR Ss regulates transcription by DNA looping. Significant transcriptional changes in the regulatory genes vicR , spxR , comE , comX , and mecA in the sptR Ss and sptS Ss mutants further indicated that SptRS Ss is part of a regulatory network that coordinates cell wall homeostasis, H 2 O 2 production, and competence. This study reveals that SptRS Ss is involved in the regulation of crucial functions for S. sanguinis persistence in the oral cavity.

Beschreibung: The hemagglutinin (H) gene of canine distemper virus (CDV) encodes the receptor-binding protein. This protein, together with the fusion (F) protein, is pivotal for infectivity since it contributes to the fusion of the viral envelope with the host cell membrane. Of the two receptors currently known for CDV (nectin-4 and the signaling lymphocyte activation molecule [SLAM]), SLAM is considered the most relevant for host susceptibility. To investigate how evolution might have impacted the host-CDV interaction, we examined the functional properties of a series of missense single nucleotide polymorphisms (SNPs) naturally accumulating within the H-gene sequences during the transition between two distinct but related strains. The two strains, a wild-type strain and a consensus strain, were part of a single continental outbreak in European wildlife and occurred in distinct geographical areas 2 years apart. The deduced amino acid sequence of the two H genes differed at 5 residues. A panel of mutants carrying all the combinations of the SNPs was obtained by site-directed mutagenesis. The selected mutant, wild type, and consensus H proteins were functionally evaluated according to their surface expression, SLAM binding, fusion protein interaction, and cell fusion efficiencies. The results highlight that the most detrimental functional effects are associated with specific sets of SNPs. Strikingly, an efficient compensational system driven by additional SNPs appears to come into play, virtually neutralizing the negative functional effects. This system seems to contribute to the maintenance of the tightly regulated function of the H-gene-encoded attachment protein. IMPORTANCE To investigate how evolution might have impacted the host-canine distemper virus (CDV) interaction, we examined the functional properties of naturally occurring single nucleotide polymorphisms (SNPs) in the hemagglutinin gene of two related but distinct strains of CDV. The hemagglutinin gene encodes the attachment protein, which is pivotal for infection. Our results show that few SNPs have a relevant detrimental impact and they generally appear in specific combinations (molecular signatures). These drastic negative changes are neutralized by compensatory mutations, which contribute to maintenance of an overall constant bioactivity of the attachment protein. This compensational mechanism might reflect the reaction of the CDV machinery to the changes occurring in the virus following antigenic variations critical for virulence.

Beschreibung: Measles and canine distemper viruses (MeV and CDV, respectively) first replicate in lymphatic and epithelial tissues by using SLAM and nectin-4 as entry receptors, respectively. The viruses may also invade the brain to establish persistent infections, triggering fatal complications, such as subacute sclerosis pan-encephalitis (SSPE) in MeV infection or chronic, multiple sclerosis-like, multifocal demyelinating lesions in the case of CDV infection. In both diseases, persistence is mediated by viral nucleocapsids that do not require packaging into particles for infectivity but are directly transmitted from cell to cell (neurons in SSPE or astrocytes in distemper encephalitis), presumably by relying on restricted microfusion events. Indeed, although morphological evidence of fusion remained undetectable, viral fusion machineries and, thus, a putative cellular receptor, were shown to contribute to persistent infections. Here, we first showed that nectin-4-dependent cell-cell fusion in Vero cells, triggered by a demyelinating CDV strain, remained extremely limited, thereby supporting a potential role of nectin-4 in mediating persistent infections in astrocytes. However, nectin-4 could not be detected in either primary cultured astrocytes or the white matter of tissue sections. In addition, a bioengineered "nectin-4-blind" recombinant CDV retained full cell-to-cell transmission efficacy in primary astrocytes. Combined with our previous report demonstrating the absence of SLAM expression in astrocytes, these findings are suggestive for the existence of a hitherto unrecognized third CDV receptor expressed by glial cells that contributes to the induction of noncytolytic cell-to-cell viral transmission in astrocytes. IMPORTANCE While persistent measles virus (MeV) infection induces SSPE in humans, persistent canine distemper virus (CDV) infection causes chronic progressive or relapsing demyelination in carnivores. Common to both central nervous system (CNS) infections is that persistence is based on noncytolytic cell-to-cell spread, which, in the case of CDV, was demonstrated to rely on functional membrane fusion machinery complexes. This inferred a mechanism where nucleocapsids are transmitted through macroscopically invisible microfusion events between infected and target cells. Here, we provide evidence that CDV induces such microfusions in a SLAM- and nectin-4-independent manner, thereby strongly suggesting the existence of a third receptor expressed in glial cells (referred to as GliaR). We propose that GliaR governs intercellular transfer of nucleocapsids and hence contributes to viral persistence in the brain and ensuing demyelinating lesions.

Beschreibung: Streptococcus mutans , a major pathogen of dental caries, may promote systemic infections after accessing the bloodstream from oral niches. In this study, we investigate pathways of complement immunity against S. mutans and show that the orphan regulator CovR (CovR Sm ) modulates susceptibility to complement opsonization and survival in blood. S. mutans blood isolates showed reduced susceptibility to C3b deposition compared to oral isolates. Reduced expression of covR Sm in blood strains was associated with increased transcription of CovR Sm -repressed genes required for S. mutans interactions with glucans ( gbpC , gbpB , and epsC ), sucrose-derived exopolysaccharides (EPS). Consistently, blood strains showed an increased capacity to bind glucan in vitro . Deletion of covR Sm in strain UA159 (UAcov) impaired C3b deposition and binding to serum IgG and C-reactive protein (CRP) as well as phagocytosis through C3b/iC3b receptors and killing by neutrophils. Opposite effects were observed in mutants of gbpC , epsC , or gtfBCD (required for glucan synthesis). C3b deposition on UA159 was abolished in C1q-depleted serum, implying that the classical pathway is essential for complement activation on S. mutans . Growth in sucrose-containing medium impaired the binding of C3b and IgG to UA159, UAcov, and blood isolates but had absent or reduced effects on C3b deposition in gtfBCD , gbpC , and epsC mutants. UAcov further showed increased ex vivo survival in human blood in an EPS-dependent way. Consistently, reduced survival was observed for the gbpC and epsC mutants. Finally, UAcov showed an increased ability to cause bacteremia in a rat model. These results reveal that CovR Sm modulates systemic virulence by regulating functions affecting S. mutans susceptibility to complement opsonization.