Antibiotic resistance is an emerging public health issue. Plasmids are one of the popular carriers to disseminate resistance genes among pathogens. However, the response of plasmid-carrying bacteria to antibiotic treatment and how these bacteria evolve to increase their resistance remain elusive. In this study, we conjugated plasmid pNDM-HK to E. coli J53 recipient cells and selected survivors using different concentrations of the broad spectrum antibiotic meropenem. After selection, transconjugants conferred varying minimum inhibitory concentrations with respect to carbapenems. We sequenced and compared the transcriptomes of transconjugants that exhibited distinct carbapenem susceptibilities, and found that the loss of outer membrane proteins led to antibiotic resistance. Moreover, we identified a novel mutation, G63S, in transcription factor OmpR which moderates the expression of outer membrane proteins. The loss of porins was due to incapability of phosphorylation, which is essential for porin transcription and carbapenem resistance. We also characterized other genes that are regulated by ompR in this mutant, which contributed to bacterial antibiotic resistance. Overall, our studies suggest antibiotic pressure after conjugation might be an alternative pathway to promote antimicrobial resistance.

The innate immune cells underlying mucosal inflammatory responses and damage during acute HIV-1 infection remain incompletely understood. Here, we report a Vδ2 subset of gut-homing γδ T cells with significantly upregulated Δ42PD1 (a PD1 isoform) in acute (~20%) HIV-1 patients compared to chronic HIV-1 patients (~11%) and healthy controls (~2%). The frequency of Δ42PD1Vδ2 cells correlates positively with plasma levels of pro-inflammatory cytokines and fatty-acid-binding protein before detectable lipopolysaccharide in acute patients. The expression of Δ42PD1 can be induced by in vitro HIV-1 infection and is accompanied by high co-expression of gut-homing receptors CCR9/CD103. To investigate the role of Δ42PD1Vδ2 cells in vivo, they were adoptively transferred into autologous humanized mice, resulting in small intestinal inflammatory damage, probably due to the interaction of Δ42PD1 with its cognate receptor Toll-like receptor 4 (TLR4). In addition, blockade of Δ42PD1 or TLR4 successfully reduced the cytokine effect induced by Δ42PD1Vδ2 cells in vitro, as well as the mucosal pathological effect in humanized mice. Our findings have therefore uncovered a Δ42PD1-TLR4 pathway exhibited by virus-induced gut-homing Vδ2 cells that may contribute to innate immune activation and intestinal pathogenesis during acute HIV-1 infection. Δ42PD1Vδ2 cells may serve as a target for the investigation of diseases with mucosal inflammation.

The ribosomal maturation factor P (RimP) is a highly conserved protein in bacteria and has been shown to be important in ribosomal assembly in Escherichia coli. Because of its central importance in bacterial metabolism, RimP represents a good potential target for drug design to combat human pathogens such as Mycobacterium tuberculosis. However, to date, the only RimP structure available is the NMR structure of the ortholog in another bacterial pathogen, Streptococcus pneumoniae. Here, we report a 2.2 Å resolution crystal structure of MSMEG_2624, the RimP ortholog in the close M. tuberculosis relative Mycobacterium smegmatis, and using in vitro binding assays, we show that MSMEG_2624 interacts with the small ribosomal protein S12, also known as RpsL. Further analyses revealed that the conserved residues in the linker region between the N-and C-terminal domains of MSMEG_2624 are essential for binding to RpsL. However, neither of the two domains alone was sufficient to form strong interactions with RpsL. More importantly, the linker region was essential for in vivo ribosomal biogenesis. Our study provides critical mechanistic insights into the role of RimP in ribosome biogenesis. We anticipate that the MSMEG_2624 crystal structure has the potential to be used for drug design to manage M. tuberculosis infections. by guest on July 10, 2020 http://www.jbc.org/ Downloaded fromFigure 4. The efficiency of in vivo ribosomal biogenesis of MSMEG_2624 mutants.A, sucrose gradient centrifugation of the WT M. smegmatis, the MSMEG_2624 knockout strain, the MSMEG_2624 knockout with the vector that expresses MSMEG_2624, and the MSMEG_2624 knockout that carries the empty vector. The vertical yellow bar marks the fraction further characterized using MS in B. B, The bar plot shows the ratio of S12/S4 in the 30S subunit of WT, the MSMEG_2624 deletion mutant strain, and the MSMEG_2624 knock-out strain with the vector that expresses MSMEG_2624. C, sucrose gradient centrifugation shows the ribosome profile of the MSMEG_2624 mutants which affects binding with RpsL (⌬P90-D93, shown in green), and which does not (P95G, shown in red). The controls of empty vector and empty vector ϩ WT are shown in cyan and purple. RimP facilitates ribosomal biogenesis by binding to S12

Gaseous biogenic amines such as putrescine, spermidine, aniline, and trimethylamine are important biomolecules that play many crucial roles in metabolism and medical diagnostics. A chemodosimetric detection assay has been developed for those gaseous amines by Ru(II)-Eu(III) heterobimetallic complexes, K{[Ru(II)((t)Bubpy)(CN)(4)](2)Eu(III)(H(2)O)(4)} (where (t)Bubpy = 4,4'-di-tert-butyl-2,2'-bipyridine). Synthesis, X-ray crystal characterization, and spectroscopic properties of this Ru(II)-Eu(III) heterobimetallic complex were reported. Binding properties of the Ru(II)-Eu(III) complex with common gases revealed that this complex is very selective to gaseous amine molecules. Sensitivity of this complex toward the amines was found as ∼log k() = 4.5-4.8. Real time monitoring of gaseous biogenic amines was applied to real fish samples (Atlantic mackerel) by studying the spectrofluorimetric responses of the Ru(II)-Eu(III) complex toward different biogenic amine concentration. GC/MS studies were also used as a reference for the studies. A linear spectrofluorimetric response was found toward biogenic amine concentration in real fish samples. This complex was found to respond specifically to those biogenic amines down to 10 ppb.