Stability of SARS-CoV-2 RNA in Nonsupplemented Saliva.

The expense of saliva collection devices designed to stabilize severe acute respiratory syndrome coronavirus 2 RNA is prohibitive to mass testing. However, virus RNA in nonsupplemented saliva is stable for extended periods and at elevated temperatures. Simple plastic tubes for saliva collection will make large-scale testing and continued surveillance easier.

Simply saliva: stability of SARS-CoV-2 detection negates the need for expensive collection devices.

Most currently approved strategies for the collection of saliva for COVID-19 diagnostics require specialized tubes containing buffers promoted for the stabilization of SARS-CoV-2 RNA and virus inactivation. Yet many of these are expensive, in limited supply, and not necessarily validated specifically for viral RNA. While saliva is a promising sample type as it can be reliably self-collected for the sensitive detection of SARS-CoV-2, the expense and availability of these collection tubes are prohibitive to mass testing efforts. Therefore, we investigated the stability of SARS-CoV-2 RNA and infectious virus detection from saliva without supplementation. We tested RNA stability over extended periods of time (2-25 days) and at temperatures representing at-home storage and elevated temperatures which might be experienced when cold chain transport may be unavailable. We found SARS-CoV-2 RNA in saliva from infected individuals is stable at 4°C, room temperature (~19°C), and 30°C for prolonged periods and found limited evidence for viral replication in saliva. This work demonstrates that expensive saliva collection options involving RNA stabilization and virus inactivation buffers are not always needed, permitting the use of cheaper collection options. Affordable testing methods are urgently needed to meet current testing demands and for continued surveillance in reopening strategies.

Itaconate is an effector of a Rab GTPase cell-autonomous host defense pathway against Salmonella.

The guanosine triphosphatase (GTPase) Rab32 coordinates a cell-intrinsic host defense mechanism that restricts the replication of intravacuolar pathogens such as Salmonella Here, we show that this mechanism requires aconitate decarboxylase 1 (IRG1), which synthesizes itaconate, a metabolite with antimicrobial activity. We find that Rab32 interacts with IRG1 on Salmonella infection and facilitates the delivery of itaconate to the Salmonella-containing vacuole. Mice defective in IRG1 rescued the virulence defect of a S. enterica serovar Typhimurium mutant specifically defective in its ability to counter the Rab32 defense mechanism. These studies provide a link between a metabolite produced in the mitochondria after stimulation of innate immune receptors and a cell-autonomous defense mechanism that restricts the replication of an intracellular bacterial pathogen.

Function, essentiality, and expression of cytochrome P450 enzymes and their cognate redox partners in Mycobacterium tuberculosis: are they drug targets?

This review covers the current knowledge of the cytochrome P450 enzymes (CYPs) of the human pathogen Mycobacterium tuberculosis (Mtb) and their endogenous redox partners, focusing on their biological function, expression, regulation, involvement in antibiotic resistance, and suitability for exploitation as antitubercular targets. The Mtb genome encodes twenty  CYPs and nine associated redox partners required for CYP catalytic activity. Transposon insertion mutagenesis studies have established the (conditional) essentiality of several of these enzymes for in vitro growth and host infection. Biochemical characterization of a handful of Mtb CYPs has revealed that they have specific physiological functions in bacterial virulence and persistence in the host. Analysis of the transcriptional response of Mtb CYPs and redox partners to external insults and to first-line antibiotics used to treat tuberculosis showed a diverse expression landscape, suggesting for some enzymes a potential role in drug resistance. Combining the knowledge about the physiological roles and expression profiles indicates that, at least five Mtb CYPs, CYP121A1, CYP125A1, CYP139A1, CYP142A1, and CYP143A1, as well as two ferredoxins, FdxA and FdxC, can be considered promising novel therapeutic targets.

Mycobacteria employ two different mechanisms to cross the blood-brain barrier.

Central nervous system (CNS) infection by Mycobacterium tuberculosis is one of the most devastating complications of tuberculosis, in particular in early childhood. In order to induce CNS infection, M. tuberculosis needs to cross specialised barriers protecting the brain. How M. tuberculosis crosses the blood-brain barrier (BBB) and enters the CNS is not well understood. Here, we use transparent zebrafish larvae and the closely related pathogen Mycobacterium marinum to answer this question. We show that in the early stages of development, mycobacteria rapidly infect brain tissue, either as free mycobacteria or within circulating macrophages. After the formation of a functionally intact BBB, the infiltration of brain tissue by infected macrophages is delayed, but not blocked, suggesting that crossing the BBB via phagocytic cells is one of the mechanisms used by mycobacteria to invade the CNS. Interestingly, depletion of phagocytic cells did not prevent M. marinum from infecting the brain tissue, indicating that free mycobacteria can independently cause brain infection. Detailed analysis showed that mycobacteria are able to cause vasculitis by extracellular outgrowth in the smaller blood vessels and by infecting endothelial cells. Importantly, we could show that this second mechanism is an active process that depends on an intact ESX-1 secretion system, which extends the role of ESX-1 secretion beyond the macrophage infection cycle.

Accelerating Early Antituberculosis Drug Discovery by Creating Mycobacterial Indicator Strains That Predict Mode of Action.

Due to the rise of drug-resistant forms of tuberculosis, there is an urgent need for novel antibiotics to effectively combat these cases and shorten treatment regimens. Recently, drug screens using whole-cell analyses have been shown to be successful. However, current high-throughput screens focus mostly on stricto sensu life/death screening that give little qualitative information. In doing so, promising compound scaffolds or nonoptimized compounds that fail to reach inhibitory concentrations are missed. To accelerate early tuberculosis (TB) drug discovery, we performed RNA sequencing on Mycobacterium tuberculosis and Mycobacterium marinum to map the stress responses that follow upon exposure to subinhibitory concentrations of antibiotics with known targets, ciprofloxacin, ethambutol, isoniazid, streptomycin, and rifampin. The resulting data set comprises the first overview of transcriptional stress responses of mycobacteria to different antibiotics. We show that antibiotics can be distinguished based on their specific transcriptional stress fingerprint. Notably, this fingerprint was more distinctive in M. marinum We decided to use this to our advantage and continue with this model organism. A selection of diverse antibiotic stress genes was used to construct stress reporters. In total, three functional reporters were constructed to respond to DNA damage, cell wall damage, and ribosomal inhibition. Subsequently, these reporter strains were used to screen a small anti-TB compound library to predict the mode of action. In doing so, we identified the putative modes of action for three novel compounds, which confirms the utility of our approach.

Cell envelope stress in mycobacteria is regulated by the novel signal transduction ATPase IniR in response to trehalose.

The cell envelope of mycobacteria is a highly unique and complex structure that is functionally equivalent to that of Gram-negative bacteria to protect the bacterial cell. Defects in the integrity or assembly of this cell envelope must be sensed to allow the induction of stress response systems. The promoter that is specifically and most strongly induced upon exposure to ethambutol and isoniazid, first line drugs that affect cell envelope biogenesis, is the iniBAC promoter. In this study, we set out to identify the regulator of the iniBAC operon in Mycobacterium marinum using an unbiased transposon mutagenesis screen in a constitutively iniBAC-expressing mutant background. We obtained multiple mutants in the mce1 locus as well as mutants in an uncharacterized putative transcriptional regulator (MMAR_0612). This latter gene was shown to function as the iniBAC regulator, as overexpression resulted in constitutive iniBAC induction, whereas a knockout mutant was unable to respond to the presence of ethambutol and isoniazid. Experiments with the M. tuberculosis homologue (Rv0339c) showed identical results. RNAseq experiments showed that this regulatory gene was exclusively involved in the regulation of the iniBAC operon. We therefore propose to name this dedicated regulator iniBAC Regulator (IniR). IniR belongs to the family of signal transduction ATPases with numerous domains, including a putative sugar-binding domain. Upon testing different sugars, we identified trehalose as an activator and metabolic cue for iniBAC activation, which could also explain the effect of the mce1 mutations. In conclusion, cell envelope stress in mycobacteria is regulated by IniR in a cascade that includes trehalose.

A fluorescence-based reporter for monitoring expression of mycobacterial cytochrome bd in response to antibacterials and during infection.

Cytochrome bd is a component of the oxidative phosphorylation pathway in many Gram-positive and Gram-negative bacteria. Next to its role as a terminal oxidase in the respiratory chain this enzyme plays an important role as a survival factor in the bacterial stress response. In Mycobacterium tuberculosis and related mycobacterial strains, cytochrome bd is an important component of the defense system against antibacterial drugs. In this report we describe and evaluate an mCherry-based fluorescent reporter for detection of cytochrome bd expression in Mycobacterium marinum. Cytochrome bd was induced by mycolic acid biosynthesis inhibitors such as isoniazid and most prominently by drugs targeting oxidative phosphorylation. We observed no induction by inhibitors of protein-, DNA- or RNA-synthesis. The constructed expression reporter was suitable for monitoring mycobacterial cytochrome bd expression during mouse macrophage infection and in a zebrafish embryo infection model when using Mycobacterium marinum. Interestingly, in both these infection models cytochrome bd levels were considerably higher than during in vitro culturing of M. marinum. The expression reporter described here can be a valuable tool for elucidating the role of cytochrome bd as a survival factor.

iniBAC induction Is Vitamin B12- and MutAB-dependent in Mycobacterium marinum.

Tuberculosis can be treated with a 6-month regimen of antibiotics. Although the targets of most of the first-line antibiotics have been identified, less research has focused on the intrabacterial stress responses that follow upon treatment with antibiotics. Studying the roles of these stress genes may lead to the identification of crucial stress-coping mechanisms that can provide additional drug targets to increase treatment efficacy. A three-gene operon with unknown function that is strongly up-regulated upon treatment with isoniazid and ethambutol is the iniBAC operon. We have reproduced these findings and show that iniBAC genes are also induced in infected host cells, although with higher variability. Next, we set out to elucidate the genetic network that results in iniBAC induction in Mycobacterium marinum By transposon mutagenesis, we identified that the operon is highly induced by mutations in genes encoding enzymes of the vitamin B12 biosynthesis pathway and the vitamin B12-dependent methylmalonyl-CoA-mutase MutAB. Lipid analysis showed that a mutA::tn mutant has decreased phthiocerol dimycocerosates levels, suggesting a link between iniBAC induction and the production of methyl-branched lipids. Moreover, a similar screen in Mycobacterium bovis BCG identified that phthiocerol dimycocerosate biosynthesis mutants cause the up-regulation of iniBAC genes. Based on these data, we propose that iniBAC is induced in response to mutations that cause defects in the biosynthesis of methyl-branched lipids. The resulting metabolic stress caused by these mutations or caused by ethambutol or isoniazid treatment may be relieved by iniBAC to increase the chance of bacterial survival.

Inorganic Phosphate Limitation Modulates Capsular Polysaccharide Composition in Mycobacteria.

Mycobacterium tuberculosis is protected by an unusual and highly impermeable cell envelope that is critically important for the successful colonization of the host. The outermost surface of this cell envelope is formed by capsular polysaccharides that play an important role in modulating the initial interactions once the bacillus enters the body. Although the bioenzymatic steps involved in the production of the capsular polysaccharides are emerging, information regarding the ability of the bacterium to modulate the composition of the capsule is still unknown. Here, we study the mechanisms involved in regulation of mycobacterial capsule biosynthesis using a high throughput screen for gene products involved in capsular α-glucan production. Utilizing this approach we identified a group of mutants that all carried mutations in the ATP-binding cassette phosphate transport locus pst These mutants collectively exhibited a strong overproduction of capsular polysaccharides, including α-glucan and arabinomannan, suggestive of a role for inorganic phosphate (Pi) metabolism in modulating capsular polysaccharide production. These findings were corroborated by the observation that growth under low Pi conditions as well as chemical activation of the stringent response induces capsule production in a number of mycobacterial species. This induction is, in part, dependent on σ factor E. Finally, we show that Mycobacterium marinum, a model organism for M. tuberculosis, encounters Pi stress during infection, which shows the relevance of our findings in vivo.

A murine monoclonal antibody to glycogen: characterization of epitope-fine specificity by saturation transfer difference (STD) NMR spectroscopy and its use in mycobacterial capsular α-glucan research.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a major pathogen responsible for 1.5 million deaths annually. This bacterium is characterized by a highly unusual and impermeable cell envelope, which plays a key role in mycobacterial survival and virulence. Although many studies have focused on the composition and functioning of the mycobacterial cell envelope, the capsular α-glucan has received relatively minor attention. Here we show that a murine monoclonal antibody (Mab) directed against glycogen cross-reacts with mycobacterial α-glucans, polymers of α(1-4)-linked glucose residues with α(1-6)-branch points. We identified the Mab epitope specificity by saturation transfer difference NMR and show that the α(1-4)-linked glucose residues are important in glucan-Mab interaction. The minimal epitope is formed by (linear) maltotriose. Notably, a Mycobacterium mutant lacking the branching enzyme GlgB does not react with the Mab; this suggests that the α(1-6)-branches form part of the epitope. These seemingly conflicting data can be explained by the fact that in the mutant the linear form of the α-glucan (amylose) is insoluble. This Mab was subsequently used to develop several techniques helpful in capsular α-glucan research. By using a capsular glucan-screening methodology based on this Mab we were able to identify several unknown genes involved in capsular α-glucan biogenesis. Additionally, we developed two methods for the detection of capsular α-glucan levels. This study therefore opens new ways to study capsular α-glucan and to identify possible targets for further research.

Rapid plasmid replicon typing by real time PCR melting curve analysis.

BACKGROUND: Genes encoding Extended Spectrum Beta Lactamases are usually located on transferable plasmids. Each plasmid contains its own replication mechanism. Carattoli et al. developed an extended PCR-based replicon typing method to characterize and identify the replicons of the major plasmid incompatibility groups in Enterobacteriaceae. Based on this method, we designed a rapid approach with amplicon detection by real-time melting curve analysis. This method appeared to be fast, sensitive, less laborious, less prone to contamination and applicable in a routine laboratory environment. RESULTS: We successfully integrated the post-PCR analysis of the replicon typing into a closed system, which leads to a 10-fold increase in sensitivity compared to agarose gel visualization. Moreover, the use of crude lysates and SYBR-green saves a considerable amount of hands-on time without decreasing the sensitivity or specificity. CONCLUSIONS: This real-time melting curve replicon typing method appears to be fast, sensitive, less laborious, less prone to contamination and applicable in a routine laboratory environment.

Chimeric HIV-1 envelope glycoproteins with potent intrinsic granulocyte-macrophage colony-stimulating factor (GM-CSF) activity.

HIV-1 acquisition can be prevented by broadly neutralizing antibodies (BrNAbs) that target the envelope glycoprotein complex (Env). An ideal vaccine should therefore be able to induce BrNAbs that can provide immunity over a prolonged period of time, but the low intrinsic immunogenicity of HIV-1 Env makes the elicitation of such BrNAbs challenging. Co-stimulatory molecules can increase the immunogenicity of Env and we have engineered a soluble chimeric Env trimer with an embedded granulocyte-macrophage colony-stimulating factor (GM-CSF) domain. This chimeric molecule induced enhanced B and helper T cell responses in mice compared to Env without GM-CSF. We studied whether we could optimize the activity of the embedded GM-CSF as well as the antigenic structure of the Env component of the chimeric molecule. We assessed the effect of truncating GM-CSF, removing glycosylation-sites in GM-CSF, and adjusting the linker length between GM-CSF and Env. One of our designed Env(GM-CSF) chimeras improved GM-CSF-dependent cell proliferation by 6-fold, reaching the same activity as soluble recombinant GM-CSF. In addition, we incorporated GM-CSF into a cleavable Env trimer and found that insertion of GM-CSF did not compromise Env cleavage, while Env cleavage did not compromise GM-CSF activity. Importantly, these optimized Env(GM-CSF) proteins were able to differentiate human monocytes into cells with a macrophage-like phenotype. Chimeric Env(GM-CSF) should be useful for improving humoral immunity against HIV-1 and these studies should inform the design of other chimeric proteins.

HIV-1 N-glycan composition governs a balance between dendritic cell-mediated viral transmission and antigen presentation.

The natural function of dendritic cells (DCs) is to capture and degrade pathogens for Ag presentation. However, HIV-1 can evade viral degradation by DCs and hijack DCs for migration to susceptible CD4(+) T lymphocytes. It is unknown what factors decide whether a virus is degraded or transmitted to T cells. The interaction of DCs with HIV-1 involves C-type lectin receptors, such as DC-specific ICAM-3-grabbing nonintegrin, which bind to the envelope glycoprotein complex (Env), which is decorated heavily with N-linked glycans. We hypothesized that the saccharide composition of the Env N-glycans is involved in avoiding viral degradation and Ag presentation, as well as preserving infectious virus for the transmission to target cells. Therefore, we studied the fate of normally glycosylated virus versus oligomannose-enriched virus in DCs. Changing the heterogeneous N-linked glycan composition of Env to uniform oligomannose N-glycans increased the affinity of HIV-1 for DC-specific ICAM-3-grabbing nonintegrin and enhanced the capture of HIV-1 by immature DCs; however, it decreased the subsequent transmission to target cells. Oligomannose-enriched HIV-1 was directed more efficiently into the endocytic pathway, resulting in enhanced viral degradation and reduced virus transfer to target cells. Furthermore, Env containing exclusively oligomannose N-glycans was presented to Env-specific CD4(+) T cells more efficiently. Taken together, our results showed that the HIV-1 N-glycan composition plays a crucial role in the balance between DC-mediated Ag degradation and presentation and DC-mediated virus transmission to target cells. This finding may have implications for the early events in HIV-1 transmission and the induction of antiviral immune responses.