Streptococcal dTDP‐L‐rhamnose biosynthesis enzymes: functional characterization and lead compound identification

Biosynthesis of the nucleotide sugar precursor dTDP‐L‐rhamnose is critical for the viability and virulence of many human pathogenic bacteria, including Streptococcus pyogenes (Group A Streptococcus; GAS), Streptococcus mutans and Mycobacterium tuberculosis. Streptococcal pathogens require dTDP‐L‐rhamnose for the production of structurally similar rhamnose polysaccharides in their cell wall. Via heterologous expression in S. mutans, we confirmed that GAS RmlB and RmlC are critical for dTDP‐L‐rhamnose biosynthesis through their action as dTDP‐glucose‐4,6‐dehydratase and dTDP‐4‐keto‐6‐deoxyglucose‐3,5‐epimerase enzymes respectively. Complementation with GAS RmlB and RmlC containing specific point mutations corroborated the conservation of previous identified catalytic residues. Bio‐layer interferometry was used to identify and confirm inhibitory lead compounds that bind to GAS dTDP‐rhamnose biosynthesis enzymes RmlB, RmlC and GacA. One of the identified compounds, Ri03, inhibited growth of GAS, other rhamnose‐dependent streptococcal pathogens as well as M. tuberculosis with an IC₅₀ of 120–410 µM. Importantly, we confirmed that Ri03 inhibited dTDP‐L‐rhamnose formation in a concentration‐dependent manner through a biochemical assay with recombinant rhamnose biosynthesis enzymes. We therefore conclude that inhibitors of dTDP‐L‐rhamnose biosynthesis, such as Ri03, affect streptococcal and mycobacterial viability and can serve as lead compounds for the development of a new class of antibiotics that targets dTDP‐rhamnose biosynthesis in pathogenic bacteria.     

Outer surface protein polymorphisms linked to host‐spirochete association in Lyme borreliae

Lyme borreliosis is caused by multiple species of the spirochete bacteria Borrelia burgdorferi sensu lato. The spirochetes are transmitted by ticks to vertebrate hosts, including small‐ and medium‐sized mammals, birds, reptiles, and humans. Strain‐to‐strain variation in host‐specific infectivity has been documented, but the molecular basis that drives this differentiation is still unclear. Spirochetes possess the ability to evade host immune responses and colonize host tissues to establish infection in vertebrate hosts. In turn, hosts have developed distinct levels of immune responses when invaded by different species/strains of Lyme borreliae. Similarly, the ability of Lyme borreliae to colonize host tissues varies among different spirochete species/strains. One potential mechanism that drives this strain‐to‐strain variation of immune evasion and colonization is the polymorphic outer surface proteins produced by Lyme borreliae. In this review, we summarize research on strain‐to‐strain variation in host competence and discuss the evidence that supports the role of spirochete‐produced protein polymorphisms in driving this variation in host specialization. Such information will provide greater insights into the adaptive mechanisms driving host and Lyme borreliae association, which will lead to the development of interventions to block pathogen spread and eventually reduce Lyme borreliosis health burden.     

Characterization of the atypical Ppz/Hal3 phosphatase system from the pathogenic fungus Cryptococcus neoformans

Ppz Ser/Thr protein phosphatases (PPases) are found only in fungi and have been proposed as potential antifungal targets. In Saccharomyces cerevisiae Ppz1 (ScPpz1) is involved in regulation of monovalent cation homeostasis. ScPpz1 is inhibited by two regulatory proteins, Hal3 and Vhs3, which have moonlighting properties, contributing to the formation of an unusual heterotrimeric PPC decarboxylase (PPCDC) complex crucial for CoA biosynthesis. Here we report the functional characterization of CnPpz1 (CNAG_03673) and two possible Hal3‐like proteins, CnHal3a (CNAG_00909) and CnHal3b (CNAG_07348) from the pathogenic fungus Cryptococcus neoformans. Deletion of CnPpz1 or CnHal3b led to phenotypes unrelated to those observed in the equivalent S. cerevisiae mutants, and the CnHal3b‐deficient strain was less virulent. CnPpz1 is a functional PPase and partially replaced endogenous ScPpz1. Both CnHal3a and CnHal3b interact with ScPpz1 and CnPpz1 in vitro but do not inhibit their phosphatase activity. Consistently, when expressed in S. cerevisiae, they poorly reproduced the Ppz1‐regulatory properties of ScHal3. In contrast, both proteins were functional monogenic PPCDCs. The CnHal3b isoform was crystallized and, for the first time, the 3D‐structure of a fungal PPCDC elucidated. Therefore, our work provides the foundations for understanding the regulation and functional role of the Ppz1‐Hal3 system in this important pathogenic fungus.     

Expression and function of the bHLH genes ALCATRAZ and SPATULA in selected Solanaceae species

The genetic mechanisms underlying fruit development have been identified in Arabidopsis and have been comparatively studied in tomato as a representative of fleshy fruits. However, comparative expression and functional analyses on the bHLH genes downstream the genetic network, ALCATRAZ (ALC) and SPATULA (SPT), which are involved in the formation of the dehiscence zone in Arabidopsis, have not been functionally studied in the Solanaceae. Here, we perform detailed expression and functional studies of ALC/SPT homologs in Nicotiana obtusifolia with capsules, and in Capsicum annuum and Solanum lycopersicum with berries. In Solanaceae, ALC and SPT genes are expressed in leaves, and all floral organs, especially in petal margins, stamens and carpels; however, their expression changes during fruit maturation according to the fruit type. Functional analyses show that downregulation of ALC/SPT genes does not have an effect on gynoecium patterning; however, they have acquired opposite roles in petal expansion and have been co‐opted in leaf pigmentation in Solanaceae. In addition, ALC/SPT genes repress lignification in time and space during fruit development in Solanaceae. Altogether, some roles of ALC and SPT genes are different between Brassicaceae and Solanaceae; while the paralogs have undergone some subfunctionalization in the former they are mostly redundant in the latter.     

Surface wax esters contribute to drought tolerance in Arabidopsis

Waxes are components of the cuticle covering the aerial organs of plants. Accumulation of waxes has previously been associated with protection against water loss, therefore contributing to drought tolerance. However, not much information is known about the function of individual wax components during water deficit. We studied the role of wax ester synthesis during drought. The wax ester load on Arabidopsis leaves and stems was increased during water deficiency. Expression of three genes, WSD1, WSD6 and WSD7 of the wax ester synthase/diacylglycerol acyltransferase (WS/DGAT or WSD) family was induced during drought, salt stress and abscisic acid treatment. WSD1 has previously been identified as the major wax ester synthase of stems. wsd1 mutants have shown reduced wax ester coverage on leaves and stems during normal or drought condition, while wax ester loads of wsd6, wsd7 and of the wsd6wsd7 double mutant were unchanged. The growth and relative water content of wsd1 plants were compromised during drought, while leaf water loss of wsd1 was increased. Enzyme assays with recombinant proteins expressed in insect cells revealed that WSD6 and WSD7 contain wax ester synthase activity, albeit with different substrate specificity compared with WSD1. WSD6 and WSD7 localize to the endoplasmic reticulum (ER)/Golgi. These results demonstrated that WSD1 is involved in the accumulation of wax esters during drought, while WSD6 and WSD7 might play other specific roles in wax ester metabolism during stress.     

Tropical rainforest flies carrying pathogens form stable associations with social nonhuman primates

Living in groups provides benefits but also incurs costs such as attracting disease vectors. For example, synanthropic flies associate with human settlements, and higher fly densities increase pathogen transmission. We investigated whether such associations also exist in highly mobile nonhuman primate (NHP) Groups. We studied flies in a group of wild sooty mangabeys (Cercocebus atys atys) and three communities of wild chimpanzees (Pan troglodytes verus) in Taï National Park, Côte d'Ivoire. We observed markedly higher fly densities within both mangabey and chimpanzee groups. Using a mark–recapture experiment, we showed that flies stayed with the sooty mangabey group for up to 12 days and for up to 1.3 km. We also tested mangabey‐associated flies for pathogens infecting mangabeys in this ecosystem, Bacillus cereus biovar anthracis (Bcbva), causing sylvatic anthrax, and Treponema pallidum pertenue, causing yaws. Flies contained treponemal (6/103) and Bcbva (7/103) DNA. We cultured Bcbva from all PCR‐positive flies, confirming bacterial viability and suggesting that this bacterium might be transmitted and disseminated by flies. Whole genome sequences of Bcbva isolates revealed a diversity of Bcbva, probably derived from several sources. We conclude that flies actively track mangabeys and carry infectious bacterial pathogens; these associations represent an understudied cost of sociality and potentially expose many social animals to a diversity of pathogens.     

Differential parameters between cytosolic 2‐Cys peroxiredoxins,PRDX1 and PRDX2

Peroxiredoxins are thiol‐dependent peroxidases that function in peroxide detoxification and H₂O₂ induced signaling. Among the six isoforms expressed in humans, PRDX1 and PRDX2 share 97% sequence similarity, 77% sequence identity including the active site, subcellular localization (cytosolic) but they hold different biological functions albeit associated with their peroxidase activity. Using recombinant human PRDX1 and PRDX2, the kinetics of oxidation and hyperoxidation with H₂O₂ and peroxynitrite were followed by intrinsic fluorescence. At pH 7.4, the peroxidatic cysteine of both isoforms reacts nearly tenfold faster with H₂O₂ than with peroxynitrite, and both reactions are orders of magnitude faster than with most protein thiols. For both isoforms, the sulfenic acids formed are in turn oxidized by H₂O₂ with rate constants of ca 2 × 10³ M^?1 s^?1 and by peroxynitrous acid significantly faster. As previously observed, a crucial difference between PRDX1 and PRDX2 is on the resolution step of the catalytic cycle, the rate of disulfide formation (11 s^?1 for PRDX1, 0.2 s^?1 for PRDX2, independent of the oxidant) which correlates with their different sensitivity to hyperoxidation. This kinetic pause opens different pathways on redox signaling for these isoforms. The longer lifetime of PRDX2 sulfenic acid allows it to react with other protein thiols to translate the signal via an intermediate mixed disulfide (involving its peroxidatic cysteine), whereas PRDX1 continues the cycle forming disulfide involving its resolving cysteine to function as a redox relay. In addition, the presence of C83 on PRDX1 imparts a difference on peroxidase activity upon peroxynitrite exposure that needs further study.     

Thermal stability of single‐domain antibodies estimated by molecular dynamics simulations

Single‐domain antibodies (sdAbs) function like regular antibodies, however, consist of only one domain. Because of their low molecular weight, sdAbs have advantages with respect to production and delivery to their targets and for applications such as antibody drugs and biosensors. Thus, sdAbs with high thermal stability are required. In this work, we chose seven sdAbs, which have a wide range of melting temperature (T_m) values and known structures. We applied molecular dynamics (MD) simulations to estimate their relative stability and compared them with the experimental data. High‐temperature MD simulations at 400 K and 500 K were executed with simulations at 300 K as a control. The fraction of native atomic contacts, Q, measured for the 400 K simulations showed a fairly good correlation with the T_m values. Interestingly, when the residues were classified by their hydrophobicity and size, the Q values of hydrophilic residues exhibited an even better correlation, suggesting that stabilization is correlated with favorable interactions of hydrophilic residues. Measuring the Q value on a per‐residue level enabled us to identify residues that contribute significantly to the instability and thus demonstrating how our analysis can be used in a mutant case study.