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Many bacteria use a cell-cell communication system called quorum sensing to coordinate population density-dependent changes in behavior. Quorum sensing involves production of and response to diffusible or secreted signals, which can vary substantially across different types of bacteria. In many species, quorum sensing modulates virulence functions and is important for pathogenesis. Over the past half-century, there has been a significant accumulation of knowledge of the molecular mechanisms, signal structures, gene regulons, and behavioral responses associated with quorum-sensing systems in diverse bacteria. More recent studies have focused on understanding quorum sensing in the context of bacterial sociality. Studies of the role of quorum sensing in cooperative and competitive microbial interactions have revealed how quorum sensing coordinates interactions both within a species and between species. Such studies of quorum sensing as a social behavior have relied on the development of “synthetic ecological” models that use nonclonal bacterial populations. In this review, we discuss some of these models and recent advances in understanding how microbes might interact with one another using quorum sensing. The knowledge gained from these lines of investigation has the potential to guide studies of microbial sociality in natural settings and the design of new medicines and therapies to treat bacterial infections.

Background/Objectives: The pulmonary administration of antibiotics can be advantageous in treating pulmonary infections by promoting high intrapulmonary drug concentrations with reduced systemic exposure. However, limited benefits have been observed for pulmonary administration versus other administration routes due to its rapid clearance from the lung. Here, the effects of structural modifications on the epithelial permeability and antibacterial potency of a third-generation cephalosporin were investigated to improve the understanding of drug properties that promote intrapulmonary retention and how they may impact efficacy. Methods: Ceftazidime was modified by attaching 18 hydrophobic, hydrophilic, and mucus-binding motifs to the carboxylic acid distant from the beta-lactam by amidation. Epithelial permeability was investigated by drug transport assays using human bronchial epithelial air–liquid interface cultures. Antibacterial potency was determined by microtiter MIC assays with B. pseudomallei, P. aeruginosa, E. coli, and S. aureus. Results: A 40–50% reduction in the transepithelial transport rate was exhibited by two PEGylated ceftazidime analogs (mPEG8- and PEG5-pyrimidin-2-amine-ceftazidime) and n-butyl-ceftazidime. An increase in the transport rate was exhibited by four analogs bearing small and hydrophobic or negatively charged motifs (n-heptane-, phenyl ethyl-, glutamic acid-, and 4-propylthiophenyl boronic acid-ceftazidime). The antibacterial potency was reduced by ≥10-fold for most ceftazidime analogs against B. pseudomallei, P. aeruginosa, and E. coli but was retained by seven ceftazidime analogs primarily bearing hydrophobic motifs against S. aureus. Conclusions: The covalent conjugation of PEGs with MW > 300 Da reduced the epithelial permeability of ceftazidime, but these modifications severely reduced antibacterial activity. To improve the pulmonary retention of antibiotics with low membrane permeability, this work suggests future molecular engineering studies to explore high-molecular-weight prodrug strategies.

Abstract Bacterial secondary metabolites play important roles in promoting survival, though few have been carefully studied in their natural context. Numerous gene clusters code for secondary metabolites in the genomes of members of the Bptm group, made up of three closely related species with distinctly different lifestyles: the opportunistic pathogen Burkholderia pseudomallei, the non-pathogenic saprophyte Burkholderia thailandensis, and the host-adapted pathogen Burkholderia mallei. Several biosynthetic gene clusters are conserved across two or all three species, and this provides an opportunity to understand how the corresponding secondary metabolites contribute to survival in different contexts in nature. In this review, we discuss three secondary metabolites from the Bptm group: bactobolin, malleilactone (and malleicyprol), and the 4-hydroxy-3-methyl-2-alkylquinolines, providing an overview of each of their biosynthetic pathways and insight into their potential ecological roles. Results of studies on these secondary metabolites provide a window into how secondary metabolites contribute to bacterial survival in different environments, from host infections to polymicrobial soil communities.

Within the bacterial genus Burkholderia, the Bptm group is made up of three closely related species: Burkholderia pseudomallei, B. thailandensis, and B. mallei. This group contains both pathogenic and non-pathogenic species each with distinctly different lifestyles. B. mallei is a host-adapted equine pathogen, B. thailandensis is a non-pathogenic soil saprophyte, and B. pseudomallei is an opportunistic human pathogen that causes the disease melioidosis, which can be fatal up to 40% of the time. The Bptm species possess many conserved biosynthetic gene clusters coding for secondary metabolites. The conservation of secondary metabolites across this group affords opportunities to learn how these metabolites are useful among each member’s diverse biological niches. Secondary metabolites can be underappreciated contributors to bacterial fitness and also have roles in pathogenicity: the regulation and chemical biology of two examples, malleilactone and methylated hydroxy-alkylquinolones (HMAQs), encoded by the mal and hmq operons, respectively, are the focus of this work.Herein, we characterize B. pseudomallei malleilactone and show that it is important for virulence in nematode hosts, as well as cytotoxic to eukaryotic cells and some Gram-positive bacteria. Studies on its regulation revealed that malleilactone can be induced by treatment with various antibiotics, notably by the antibiotic trimethoprim via the activator MalR, and that a global secondary metabolite regulator ScmR, which is activated by acyl-homoserine lactone-dependent quorum sensing, strongly suppresses malleilactone production in later growth stages, likely by blocking its activation by MalR.Further contained within this work are the novel findings that efflux is important for malleilactone export and for mitigating malleilactone self-toxicity. We show that the B. pseudomallei BpeEF-OprC efflux pump system, encoded adjacent to the mal gene cluster, exports endogenous malleilactone into the extracellular environment. Notably, our results show that BpeEF-OprC protects mal-expressing cells from self-toxic effects of mal biosynthesis, especially under oxidative stress and in conditions where the recently-described malleicyprol-family malleilactone isomers, which were recently reported in B. thailandensis to have increased toxicity relative to malleilactone, are likely favored. We demonstrate that BpeT and BpeS, known regulators of the bpeEF-oprC operon, also play minor roles in mal cluster regulation. Additionally, studies within reveal that the mal genes are strongly upregulated under iron starvation conditions, in which we see that malleilactone provides a fitness benefit to wild-type cells, though in a siderophore-dependent manner. Our B. pseudomallei-purified malleilactone can bind to ferric iron. Taken all together, these findings all point to a notable and previously under-characterized contribution of malleilactone to B. pseudomallei fitness in situations of iron limitation. The new evidence from this dissertation suggests that malleilactone promotes fitness under stress, including growth- inhibiting antibiotics, host immune responses, and nutrient (iron) limitation, and that B. pseudomallei possesses an innate mal resistance mechanism.Regarding the HMAQ-family compounds, a transposon mutant screen performed by collaborators helped to identify HMAQs as B. thailandensis-produced antimicrobial compounds. Our results demonstrate using both liquid and plate co-culture assays that HMAQs are important for B. thailandensis to compete with another soil species, Bacillus subtilis. We also show that previously-uncharacterized HmqL catalyzes the formation of N-oxide derivatives of HMAQs in B. thailandensis, which have further increased antimicrobial potency against B. subtilis.Altogether, in this work we provide new insights into the biology of B. thailandensis and B. pseudomallei malleilactone and HMAQ secondary metabolites and their roles in survival in the host and in polymicrobial soil communities, and information that could be important for developing new therapeutics to treat melioidosis and other diseases.

The bacterium Burkholderia thailandensis produces an arsenal of secondary metabolites that have diverse structures and roles in the ecology of this soil-dwelling bacterium. In liquid co-culture experiments, B. thailandensis secretes an antimicrobial that nearly eliminates another soil bacterium, Bacillus subtilis. To identify the antimicrobial, we used a transposon mutagenesis approach. This screen identified antimicrobial-defective mutants with insertions in the hmqA, hmqC and hmqF genes involved in biosynthesis of a family of 2-alkyl-4(1H)-quinolones called 4-hydroxy-3-methyl-2-alkenylquinolines (HMAQs), which are closely related to the Pseudomonas aeruginosa 4-hydroxy-2-alkylquinolines (HAQs). Insertions also occurred in the previously uncharacterized gene BTH_II1576. Results confirm that BTH_II1576 is involved in generating N-oxide derivatives of HMAQs (HMAQ-NO) in B. thailandensis and that HMAQ-NOs are sufficient to eliminate B. subtilis in co-cultures. Moreover, synthetic HMAQ-NO is ∼50-fold more active than HMAQ. Both the methyl group and the length of the carbon side chain account for high activity of HMAQ-NO against B. subtilis. The results provide new information on the biosynthesis and activities of HMAQs and reveal new insight into how these molecules might be important for the ecology of B. thailandensis. The soil bacterium Burkholderia thailandensis produces 2-alkyl-4(1H)-quinolones, mostly methylated 4-hydroxy-alkenylquinolines, a family of relatively unstudied metabolites similar to molecules also synthesized by Pseudomonas aeruginosa. Several of the methylated 4-hydroxy-alkenylquinolines have antimicrobial activity against other species. We show that N-oxidated methyl-alkenylquinolines are particularly antimicrobial and sufficient to kill Bacillus subtilis in co-cultures. We confirmed their biosynthesis requires the previously unstudied protein HmqL. These results provide new information about the biology of 2-alkyl-4(1H)-quinolones, particularly the methylated 4-hydroxy-alkenylquinolines, which are unique to B. thailandensis. This study also has importance for understanding B. thailandensis secondary metabolites and has implications for potential therapeutic development.

The bacterium Burkholderia thailandensis produces an arsenal of secondary metabolites that have diverse structures and roles in the ecology of this soil-dwelling bacterium. In liquid co-culture experiments, B. thailandensis secretes an antimicrobial that nearly eliminates another soil bacterium, Bacillus subtilis. To identify the antimicrobial, we used a transposon mutagenesis approach. This screen identified antimicrobial-defective mutants with insertions in the hmqA, hmqC and hmqF genes involved in biosynthesis of a family of 2-alkyl-4(1H)-quinolones called 4-hydroxy-3-methyl-2-alkenylquinolines (HMAQs), which are closely related to the Pseudomonas aeruginosa 4-hydroxy-2-alkylquinolines (HAQs). Insertions also occurred in the previously uncharacterized gene BTH_II1576. Results confirm that BTH_II1576 is involved in generating N-oxide derivatives of HMAQs (HMAQ-NO) in B. thailandensis and that HMAQ-NOs are sufficient to eliminate B. subtilis in co-cultures. Moreover, synthetic HMAQ-NO is ~50-fold more active than HMAQ. Both the methyl group and the length of the carbon side chain account for high activity of HMAQ-NO against B. subtilis. The results provide new information on the biosynthesis and activities of HMAQs and reveal new insight into how these molecules might be important for the ecology of B. thailandensis. Competing Interest Statement The authors have declared no competing interest.

To the Editor: Heparin is commonly used in newborn intensive care units to maintain the patency of umbilical-artery catheters, and Lesko et al. (May 1 issue)* now report an association between such heparin use and intraventricular hemorrhage in low-birth-weight infants. But the published paper does not provide the analysis that would securely separate the effect of heparin on the incidence of intraventricular hemorrhage from the effect of umbilical catheter placement itself. The marked differences in birth weight and degree of illness between the cases and controls make it likely that the cases were more often exposed to catheterization. To establish . . . No extract is available for articles shorter than 400 words.

Relatlimab and nivolumab combination immunotherapy improves progression-free survival over nivolumab monotherapy in patients with unresectable advanced melanoma 1 . We investigated this regimen in patients with resectable clinical stage III or oligometastatic stage IV melanoma (NCT02519322). Patients received two neoadjuvant doses (nivolumab 480 mg and relatlimab 160 mg intravenously every 4 weeks) followed by surgery, and then ten doses of adjuvant combination therapy. The primary end point was pathologic complete response (pCR) rate 2 . The combination resulted in 57% pCR rate and 70% overall pathologic response rate among 30 patients treated. The radiographic response rate using Response Evaluation Criteria in Solid Tumors 1.1 was 57%. No grade 3–4 immune-related adverse events were observed in the neoadjuvant setting. The 1- and 2-year recurrence-free survival rate was 100% and 92% for patients with any pathologic response, compared to 88% and 55% for patients who did not have a pathologic response ( P  = 0.005). Increased immune cell infiltration at baseline, and decrease in M2 macrophages during treatment, were associated with pathologic response. Our results indicate that neoadjuvant relatlimab and nivolumab induces a high pCR rate. Safety during neoadjuvant therapy is favourable compared to other combination immunotherapy regimens. These data, in combination with the results of the RELATIVITY-047 trial 1 , provide further confirmation of the efficacy and safety of this new immunotherapy regimen. Patients with resectable clinical stage III or oligometastatic stage IV melanoma were given neoadjuvant relatlimab and nivolumab combination immunotherapy, which induced a high pathologic complete response rate, indicating the efficacy and safety of this regimen.