ACS Infectious Diseases最新文献

Secondary messengers are small, diffusible signaling molecules that transmit information from environmental cues detected at the cell surface by extracellular signaling molecules (primary messengers) to effector proteins, thereby enabling an appropriate cellular response. These molecules include cyclic nucleotides, alarmones, and lipid-derived metabolites and are ubiquitous regulators, influencing processes such as growth, metabolism, and neurotransmission in mammalian cells, as well as chemotaxis, biofilm formation, and metabolism in prokaryotes. Mycobacterium tuberculosis encodes an extensive array of genes dedicated to the synthesis and degradation of a diverse range of secondary messenger molecules. Given its highly intricate intracellular lifestyle and its ability to endure and persist in hostile and fluctuating environments, there is significant potential for crosstalk between host and bacterial secondary messengers. M. tuberculosis has likely co-opted these signaling processes within the host cell to facilitate its own pathogenesis and virulence. Recent studies have begun to elucidate the complex and multifaceted roles played by some of these secondary messengers, highlighting their capacity to regulate mycobacterial physiology while simultaneously modulating host immune responses. This review summarizes the current understanding of secondary messenger signaling in M. tuberculosis and explores how this knowledge is being leveraged to develop improved vaccines and therapeutic strategies.

Based on sequencing data, mutations at rpoB codon 491 ofMycobacterium tuberculosisare associated with rifampicin resistance, but current commercial and WHO-endorsed genotypic tests fail to detect them. As a result, resistant infections go untreated, driving transmission and multidrug resistance. A real-time PCR assay by André et al. specifically screens for I491F but omits other codon 491 mutations. To address this gap, a single-sample screening method using asymmetric PCR followed by melt analysis was developed for the three sequence-identified variants, I491F/N/M. Each sample contained a melt probe matching the susceptible sequence, which, after asymmetric PCR spanning codon 491, hybridized with the excess strand to form a duplex. The duplex's melt temperature (T_m) was then measured. To enable single-sample classification, each reaction also included double-stranded L-DNA identical to the probe and wild-type PCR product duplex. Susceptibility was determined by the within-sample T_m difference between the probe-product and L-DNA duplexes. The approach was evaluated and compared to the André assay across two calibrated PCR instruments using synthetic rpoB wild-type and variant sequences. As expected, the André assay distinguished wild-type from I491F samples but misclassified I491N and I491M samples based on multisample T_m comparison. In contrast, our single-sample classification strategy used within-sample T_m differences, classifying samples as rifampicin-susceptible when the within-sample T_m difference was less than 0.83 °C. With this approach, the method achieved 100% sensitivity and 100% specificity across both PCR instruments. Although demonstrated for rpoB codon 491, this assay design is readily adaptable to any other sequence-identified, clinically significant mutation hotspot.

The altered tropism and infection severity of the evolved SARS-CoV-2 variants indicate engagement of attachment factors other than the ACE2 receptor for the cellular attachment and entry of the virus. In this work, we report the binding of Omicron, Delta, and B.1.1.8 (A2a type) variants to gangliosides (GD1a, GM3, GM1) with terminal sialic acid (SA). The binding kinetics of intact virus particles to these ganglioside-embedded lipid membranes reveal that the affinity of Omicron for GD1a (two SA residues) is the highest, and the lowest affinity is that of B.1.1.8 for GM1 (one SA at the branched chain). Our TIRF imaging data confirm that SA and acetylated SA can inhibit the virus attachment to the bilayers but at millimolar concentration. We evaluated tetravalent glycoclusters, i.e., sialo-porphyrin, galactose-porphyrin, and glucose-porphyrin, as multivalent inhibitors of SARS-CoV-2. Our results show that membrane attachment of the variants is blocked by the micromolar concentration of sialo-porphyrin. Even the glycocluster effectively inhibits cellular infection caused by the variants.

An estimated 1.5 billion people worldwide are infected with at least one parasitic nematode species classified as soil-transmitted helminths (STHs). The recommended control strategy is to reduce morbidity using a single oral dose of the benzimidazole drugs, albendazole and mebendazole. The extensive use of benzimidazoles over the last decades has increased the risk of emerging drug resistance. Additional drawbacks, such as insufficient drug efficacy, particularly against hookworm and whipworm infections, highlight the urgent need for new and improved treatment options. In this work, we present the synthesis, characterization, and biological evaluation of four novel (organometallic and benzyl) derivatives (1-4) of the broad-spectrum anthelmintic thiabendazole. The in vitro evaluation of the derivatives on different life stages of five nematode species and Schistosoma mansoni demonstrated that the activity profile of thiabendazole could be extended. The highest activity in vitro was observed with benzyl derivative 2 against adult Trichuris muris (80% activity at 100 μM, after 72 h) compared to the parent compound thiabendazole (15% activity). Both ferrocenyl (1 and 3) and ruthenocenyl (4) derivatives demonstrated notable efficacy against adult S. mansoni at 50 μM. No toxicity was seen using the hepatocyte-derived carcinoma cell line HUH7 and the human neuroblastoma cell line SH-SY5Y. In vivo studies in the Heligmosomoides polygyrus mouse model revealed worm burden reductions of 61-78% following single oral doses of 100-200 mg/kg. Future derivatization efforts could focus on two separate targets: one aimed at enhancing STH activity and a second series pursuing the antischistosomal activity.

The German cockroach, Blattella germanica, is a widespread indoor pest and a vector of enteric human pathogens, including Salmonella enterica serovar Typhimurium (S. Typhimurium). Insecticidal baits are the most commonly used tools to control these cockroaches in built environments. Sublethal exposure to insecticidal baits has been a major driver of adaptive evolution, leading to physiological resistance to insecticides and behavioral aversion to glucose in some cockroach populations. Here, we conducted the first study investigating the effects of sublethal bait exposure on human pathogen biology in B. germanica. Our results show that a sublethal exposure to bait containing the common insecticide indoxacarb can increase susceptibility to subsequent infection by ingested S. Typhimurium in surviving cockroaches within the same generation. Interestingly, increased susceptibility to infection after sublethal bait exposure was cockroach strain dependent and did not increase the rate of shedding of the pathogen in excreta. These findings establish for the first time a potential link between a common anthropogenic intervention used to control this prevalent indoor pest and its capacity to maintain pathogens. In doing so, our work reveals a possible unintended consequence of failed pest control efforts. That is, some cockroach populations may become inadvertently more adept at maintaining pathogens due to sublethal exposure to baits stemming from existing insecticide resistance. Additional studies should further investigate this phenomenon to determine its extent and impact.

Antimicrobial resistance (AMR) is one of the most pressing global health threats, urgently requiring new classes of antibiotics with differentiated mechanisms of action (MOA). Conjugated oligoelectrolytes (COEs) represent a molecular platform for designing antimicrobial agents structurally distinct from commercially available drugs. However, questions remain regarding their MOA. Herein, we show that COE treatment causes distinct phenotypes from well-established membrane-active antibiotics, with differences arising from structural variations, such as pendant group hydrophobicity. This was revealed through bacterial cytological profiling approaches, single-cell quantitative morphological analysis, and dye localization following treatment against Gram-negative (Escherichia coli) and Gram-positive (Bacillus subtilis) bacteria. E. coli treatment with PNH2 and 1B resulted in micrometer-sized membrane vesicles, which are absent in 2-2H-treated cells. COE-treated B. subtilis featured overproduction of regions of increased fluidity (RIFs), relative to untreated cells. In contrast to the originally postulated membrane pinching mechanism, these findings support a MOA for COEs that relies predominantly on membrane restructuring, thereby providing new guidelines for further COE-based antibiotic design.

Pks13, an essential enzyme for Mycobacterium tuberculosis (Mtb) cell wall biosynthesis, represents a promising target for antimicrobial intervention. Previously, the benzofuran derivative TAM16 was identified as a potent inhibitor of Pks13 through interaction with the thioesterase (TE) domain, but its development was halted due to cardiotoxicity. Therefore, we sought to identify an alternative scaffold that demonstrated good whole-cell activity that we demonstrate had a mode of action (MOA) similar to that of TAM16. To achieve this, we employed a scaffold hopping approach, leading to the discovery of a benzoxazole (BZX) scaffold that was determined to target the Pks13 TE domain. We then explored various structure-activity relationship (SAR) studies of the series, which resulted in the identification of a prototype BZX lead. Several of the novel BZX compounds showed potent minimum inhibitory concentrations (MICs) against Mtb and low to no toxicity in cytotoxicity assays. These compounds showed on-target activity, as evidenced by the induction of the BCG iniBAC cell wall damage reporter, inhibition of mycolic acid synthesis, and resistance mutations mapping to the TE domain of Pks13 in Mycobacterium smegmatis (Msm). Overall, we believe that the BZX scaffold represents a new and promising structural class with high potential to advance antitubercular drug discovery.

Mycobacterium avium subsp. paratuberculosis (Map) causes Johne's disease (JD), a chronic infection responsible for considerable economic losses to dairy industries worldwide. Genetically clonal, Map has evolved into three distinct genetic lineages designated CII, for bovine strains, and SI and SIII, for ovine strains. Previous studies have established that Map does not produce glycopeptidolipids, characteristic of the cell wall surface of mycobacteria belonging to the M. avium complex, but rather sugar-free lipopeptide compounds synthesized by nonribosomal peptide synthetases. In this study, we combined genomic, machine learning, (bio)chemical, and analytical approaches to identify the metabolites biosynthesized by NRPS in the most ancestral SI strains of Map. We thus characterized a lipotripeptide (L3P-2) signature for the SI genetic lineage, demonstrating that the evolution of this Map subspecies has been accompanied by a diversification of the cell wall lipopeptides. Finally, L3P-2 shows promise for improved serological diagnosis of JD.

Malaria, toxoplasmosis, and cryptosporidiosis are caused by apicomplexan parasites Plasmodium spp., Toxoplasma gondii, and Cryptosporidium parvum, respectively, and pose major health challenges. Their therapies are inadequate, ineffective or threatened by drug resistance. The development of novel drugs against them requires innovative and resource-efficient strategies. We exploited the kinome conservation of these parasites to determine the cellular targets and effects of two Plasmodium falciparum inhibitors in T. gondii and C. parvum. The imidazoles, (R)-RY-1-165 and (R)-RY-1-185, were developed to target the cGMP dependent protein kinase of P. falciparum (PfPKG), orthologs of which are present in T. gondii and C. parvum. Using structural and modeling approaches we determined that the molecules bind stereospecifically and interact with PfPKG in a manner unique among described inhibitors. We used enzymatic assays and mutant P. falciparum expressing PfPKG with a substituted "gatekeeper" residue to determine that cellular activity of the molecules is mediated through targets additional to PfPKG. These likely include P. falciparum calcium dependent protein kinase 1 and 4 (PfCDPK-1, -4), kinases that, like PfPKG, have small amino acids at the "gatekeeper" position. The molecules are active against T. gondii and C. parvum, with T. gondii tachyzoites being particularly sensitive. Using mutant parasites, enzyme assays and modeling studies we demonstrate that targets in T. gondii include TgPKG, TgCDPK1, TgCDPK4 and the mitogen activated kinase-like 1 (MAPKL-1). Our results suggest that this scaffold holds promise for the development of new toxoplasmosis drugs.

The main protease (MPro) of SARS-CoV-2 is a critical enzyme required for viral replication, making it a prime target for antiviral drug development. Covalent inhibitors, which form a stable interaction with the catalytic C145, have demonstrated strong inhibition of MPro, but the influence of steric and electronic properties of P2 substituents, designed to engage the S2 substrate-binding subsite within the MPro active site, on inhibitor binding affinity remains underexplored. In this study, we design and characterize two hybrid covalent inhibitors, BBH-3 and BBH-4, and present their X-ray crystallographic structures in complex with MPro, providing molecular insights into how their distinct P2 groups, a dichlorobenzyl moiety in BBH-3 and an adamantyl substituent in BBH-4, affect binding conformation and active site adaptability. Comparative structural analyses with previously characterized inhibitors, including BBH-2 and Mcule-5948770040, reveal how the P2 bulkiness and electronic properties influence active site dynamics, particularly through interactions with the S2 and S5 subsites. The P2 group of BBH-3 induces conformational shifts in the S2 helix and the S5 loop, while BBH-4 displaces M49, stabilizing its binding through hydrophobic interactions. Isothermal titration calorimetry further elucidates the impact of P2 modifications on inhibitor affinity, revealing a delicate balance between enthalpic and entropic contributions. The data demonstrate that BBH-3 exhibits less favorable binding, affirming that dichlorobenzyl substitution at the P2 position has a more negative impact on the affinity for MPro than bulky saturated cyclic groups. This underscores the feature that MPro active site malleability may be accompanied by a conformational strain.