Advancing the fitness of gut commensal bacteria.

Nutrient starvation of beneficial bacteria helps them colonize the human gut.

Bacteria require phase separation for fitness in the mammalian gut.

Therapeutic manipulation of the gut microbiota holds great potential for human health. The mechanisms bacteria use to colonize the gut therefore present valuable targets for clinical intervention. We now report that bacteria use phase separation to enhance fitness in the mammalian gut. We establish that the intrinsically disordered region (IDR) of the broadly and highly conserved transcription termination factor Rho is necessary and sufficient for phase separation in vivo and in vitro in the human commensal Bacteroides thetaiotaomicron. Phase separation increases transcription termination by Rho in an IDR-dependent manner. Moreover, the IDR is critical for gene regulation in the gut. Our findings expose phase separation as vital for host-commensal bacteria interactions and relevant for novel clinical applications.

Control of Bacterial Virulence through the Peptide Signature of the Habitat.

To optimize fitness, pathogens selectively activate their virulence program upon host entry. Here, we report that the facultative intracellular bacterium Listeria monocytogenes exploits exogenous oligopeptides, a ubiquitous organic N source, to sense the environment and control the activity of its virulence transcriptional activator, PrfA. Using a genetic screen in adsorbent-treated (PrfA-inducing) medium, we found that PrfA is functionally regulated by the balance between activating and inhibitory nutritional peptides scavenged via the Opp transport system. Activating peptides provide essential cysteine precursor for the PrfA-inducing cofactor glutathione (GSH). Non-cysteine-containing peptides cause promiscuous PrfA inhibition. Biophysical and co-crystallization studies reveal that peptides inhibit PrfA through steric blockade of the GSH binding site, a regulation mechanism directly linking bacterial virulence and metabolism. L. monocytogenes mutant analysis in macrophages and our functional data support a model in which changes in the balance of antagonistic Opp-imported oligopeptides promote PrfA induction intracellularly and PrfA repression outside the host.

Listeria Placental Infection.

The Gram-positive facultative intracellular bacterium Listeria monocytogenes is the causative agent of listeriosis, a severe food-borne infection. Pregnant women are at risk of contracting listeriosis, which can potentially lead to miscarriage, stillbirth, preterm birth, and congenital neonatal infections. While other systemic bacterial infections may result in adverse pregnancy outcomes at comparable frequencies, L. monocytogenes has particular notoriety because fetal complications largely occur in the absence of overt illness in the mother, delaying medical intervention. Here, we briefly review the pathophysiology and mechanisms of maternofetal listeriosis, discussed in light of a recent mBio report on Listeria transplacental infection in a nonhuman primate model.

Design and synthesis of purine analogues as highly specific ligands for FcyB, a ubiquitous fungal nucleobase transporter.

In the course of our study on fungal purine transporters, a number of new 3-deazapurine analogues have been rationally designed, based on the interaction of purine substrates with the Aspergillus nidulans FcyB carrier, and synthesized following an effective synthetic procedure. Certain derivatives have been found to specifically inhibit FcyB-mediated [3H]-adenine uptake. Molecular simulations have been performed, suggesting that all active compounds interact with FcyB through the formation of hydrogen bonds with Asn163, while the insertion of hydrophobic fragments at position 9 and N6 of 3-deazaadenine enhanced the inhibition.

Functional characterization of NAT/NCS2 proteins of Aspergillus brasiliensis reveals a genuine xanthine-uric acid transporter and an intrinsically misfolded polypeptide.

The Nucleobase-Ascorbate Transporter (NAT) family includes members in nearly all domains of life. Functionally characterized NAT transporters from bacteria, fungi, plants and mammals are ion-coupled symporters specific for the uptake of purines, pyrimidines and related analogues. The characterized mammalian NATs are specific for the uptake of L-ascorbic acid. In this work we identify in silico a group of fungal putative transporters, named UapD-like proteins, which represent a novel NAT subfamily. To understand the function and specificity of UapD proteins, we cloned and functionally characterized the two Aspergillus brasiliensis NAT members (named AbUapC and AbUapD) by heterologous expression in Aspergillus nidulans. AbUapC represents canonical NATs (UapC or UapA), while AbUapD represents the new subfamily. AbUapC is a high-affinity, high-capacity, H(+)/xanthine-uric acid transporter, which can also recognize other purines with very low affinity. No apparent transport function could be detected for AbUapD. GFP-tagging showed that, unlike AbUapC which is localized in the plasma membrane, AbUapD is ER-retained and degraded in the vacuoles, a characteristic of misfolded proteins. Chimeric UapA/AbUapD molecules are also turned-over in the vacuole, suggesting that UapD includes intrinsic peptidic sequences leading to misfolding. The possible evolutionary implication of such conserved, but inactive proteins is discussed.

Origin, diversification and substrate specificity in the family of NCS1/FUR transporters.

NCS1 proteins are H(+)/Na(+) symporters specific for the uptake of purines, pyrimidines and related metabolites. In this article, we study the origin, diversification and substrate specificity of fungal NCS1 transporters. We show that the two fungal NCS1 sub-families, Fur and Fcy, and plant homologues originate through independent horizontal transfers from prokaryotes and that expansion by gene duplication led to the functional diversification of fungal NCS1. We characterised all Fur proteins of the model fungus Aspergillus nidulans and discovered novel functions and specificities. Homology modelling, substrate docking, molecular dynamics and systematic mutational analysis in three Fur transporters with distinct specificities identified residues critical for function and specificity, located within a major substrate binding site, in transmembrane segments TMS1, TMS3, TMS6 and TMS8. Most importantly, we predict and confirm that residues determining substrate specificity are located not only in the major substrate binding site, but also in a putative outward-facing selective gate. Our evolutionary and structure-function analysis contributes in the understanding of the molecular mechanisms underlying the functional diversification of eukaryotic NCS1 transporters, and in particular, forward the concept that selective channel-like gates might contribute to substrate specificity.

Modelling, substrate docking and mutational analysis identify residues essential for function and specificity of the major fungal purine transporter AzgA.

The AzgA purine/H(+) symporter of Aspergillus nidulans is the founding member of a functionally and phylogenetically distinct transporter family present in fungi, bacteria and plants. Here a valid AzgA topological model is built based on the crystal structure of the Escherichia coli uracil transporter UraA, a member of the nucleobase-ascorbate transporter (NAT/NCS2) family. The model consists of 14 transmembrane, mostly α-helical, segments (TMSs) and cytoplasmic N- and C-tails. A distinct compact core of 8 TMSs, made of two intertwined inverted repeats (TMSs 1-4 and 8-11), is topologically distinct from a flexible domain (TMSs 5-7 and 12-14). A putative substrate binding cavity is visible between the core and the gate domains. Substrate docking, molecular dynamics and mutational analysis identified several residues critical for purine binding and/or transport in TMS3, TMS8 and TMS10. Among these, Asn131 (TMS3), Asp339 (TMS8) and Glu394 (TMS10) are proposed to directly interact with substrates, while Asp342 (TMS8) might be involved in subsequent substrate translocation, through H(+) binding and symport. Thus, AzgA and other NAT transporters use topologically similar TMSs and amino acid residues for substrate binding and transport, which in turn implies that AzgA-like proteins constitute a distant subgroup of the ubiquitous NAT family.

Transport assays in filamentous fungi: kinetic characterization of the UapC purine transporter of Aspergillus nidulans.

Transport assays allow the direct kinetic analysis of a specific transporter by measuring apparent Km and Vmax values, and permit the characterization of substrate specificity profiles through competition assays. In this protocol we describe a rapid and easy method for performing uptake assays in the model filamentous ascomycete Aspergillus nidulans. Our method makes use of A. nidulans germinating conidiospores at a defined morphological stage in which most transporters show maximal expression, avoiding technical difficulties associated with the use of mycelia. In combination with the ease of construction of genetic null mutants in A. nidulans, our method allows the rigorous characterization of any transporter in genetic backgrounds that are devoid of other transporters of similar specificity. Here, we use this method to characterize the kinetic parameters and the specificity profile of UapC, a uric acid-xanthine transporter present in all ascomycetes and member of the ubiquitous Nucleobase-Ascorbate Transporter family, in specific genetic backgrounds lacking other relevant transporters.

Modeling, substrate docking, and mutational analysis identify residues essential for the function and specificity of a eukaryotic purine-cytosine NCS1 transporter.

The recent elucidation of crystal structures of a bacterial member of the NCS1 family, the Mhp1 benzyl-hydantoin permease from Microbacterium liquefaciens, allowed us to construct and validate a three-dimensional model of the Aspergillus nidulans purine-cytosine/H(+) FcyB symporter. The model consists of 12 transmembrane α-helical, segments (TMSs) and cytoplasmic N- and C-tails. A distinct core of 10 TMSs is made of two intertwined inverted repeats (TMS1-5 and TMS6-10) that are followed by two additional TMSs. TMS1, TMS3, TMS6, and TMS8 form an open cavity that is predicted to host the substrate binding site. Based on primary sequence alignment, three-dimensional topology, and substrate docking, we identified five residues as potentially essential for substrate binding in FcyB; Ser-85 (TMS1), Trp-159, Asn-163 (TMS3), Trp-259 (TMS6), and Asn-354 (TMS8). To validate the role of these and other putatively critical residues, we performed a systematic functional analysis of relevant mutants. We show that the proposed substrate binding residues, plus Asn-350, Asn-351, and Pro-353 are irreplaceable for FcyB function. Among these residues, Ser-85, Asn-163, Asn-350, Asn-351, and Asn-354 are critical for determining the substrate binding affinity and/or the specificity of FcyB. Our results suggest that Ser-85, Asn-163, and Asn-354 directly interact with substrates, Trp-159 and Trp-259 stabilize binding through π-π stacking interactions, and Pro-353 affects the local architecture of substrate binding site, whereas Asn-350 and Asn-351 probably affect substrate binding indirectly. Our work is the first systematic approach to address structure-function-specificity relationships in a eukaryotic member of NCS1 family by combining genetic and computational approaches.