Mycobacterium tuberculosis H37Rv Strain Increases the Frequency of CD3+TCR+ Macrophages and Affects Their Phenotype, but Not Their Migration Ability

In mycobacterial infections, the number of cells from two newly discovered subpopulations of CD3+ myeloid cells are increased at the infection site; one type expresses the T cell receptor (CD3+TCRαβ+) and the other does not (CD3+TCRαβ−). The role of Mycobacterium tuberculosis (Mtb) virulence in generating these subpopulations and the ability of these cells to migrate remains unclear. In this study, monocyte-derived macrophages (MDMs) infected in vitro with either a virulent (H37Rv) or an avirulent (H37Ra) Mtb strain were phenotypically characterized based on three MDM phenotypes (CD3−, CD3+TCRαβ+, and CD3+TCRαβ−); then, their migration ability upon Mtb infection was evaluated. We found no differences in the frequency of CD3+ MDMs at 24 h of infection with either Mtb strain. However, H37Rv infection increased the frequency of CD3+TCRαβ+ MDMs at a multiplicity of infection of 1 and altered the expression of CD1b, CD1c, and TNF on the surface of cells from both the CD3+ MDM subpopulations; it also modified the expression of CCR2, CXCR1, and CCR7, thus affecting CCL2 and IL-8 levels. Moreover, H37Rv infection decreased the migration ability of the CD3− MDMs, but not CD3+ MDMs. These results confirm that the CD3+ macrophage subpopulations express chemokine receptors that respond to chemoattractants, facilitating cell migration. Together, these data suggest that CD3+ MDMs are a functional subpopulation involved in the immune response against Mtb.

Steroid Metabolism in Thermophilic Actinobacterium Saccharopolyspora hirsuta VKM Ac-666T

The application of thermophilic microorganisms opens new prospects in steroid biotechnology, but little is known to date on steroid catabolism by thermophilic strains. The thermophilic strain Saccharopolyspora hirsuta VKM Ac-666T has been shown to convert various steroids and to fully degrade cholesterol. Cholest-4-en-3-one, cholesta-1,4-dien-3-one, 26-hydroxycholest-4-en-3-one, 3-oxo-cholest-4-en-26-oic acid, 3-oxo-cholesta-1,4-dien-26-oic acid, 26-hydroxycholesterol, 3β-hydroxy-cholest-5-en-26-oic acid were identified as intermediates in cholesterol oxidation. The structures were confirmed by 1H and 13C-NMR analyses. Aliphatic side chain hydroxylation at C26 and the A-ring modification at C3, which are putatively catalyzed by cytochrome P450 monooxygenase CYP125 and cholesterol oxidase, respectively, occur simultaneously in the strain and are followed by cascade reactions of aliphatic sidechain degradation and steroid core destruction via the known 9(10)-seco-pathway. The genes putatively related to the sterol and bile acid degradation pathways form three major clusters in the S. hirsuta genome. The sets of the genes include the orthologs of those involved in steroid catabolism in Mycobacterium tuberculosis H37Rv and Rhodococcus jostii RHA1 and related actinobacteria. Bioinformatics analysis of 52 publicly available genomes of thermophilic bacteria revealed only seven candidate strains that possess the key genes related to the 9(10)-seco pathway of steroid degradation, thus demonstrating that the ability to degrade steroids is not widespread among thermophilic bacteria.

In Vitro Interactions of Costus speciosus (J. Koenig) Sm., Cymbopogon citratus (Dc. Ex Nees) Stapf. and Tabernaemontana coronaria (L.) Willd. with First-Line Anti-Tuberculosis Drugs Against Mycobacterium tuberculosis H37rv

Global tuberculosis (TB) burden underscores the importance of developing new effective anti-TB drugs. This study was concerned with prospecting for potential anti-TB agents from Malaysian medicinal plants. In our previous study, we have reported that n-hexane fractions of Costus speciosus (C. speciosus) (J. Koening) Sm., Cymbopogon citratus (C. citratus ) (DC.) Stapf. and Tabernaemontana coronaria (T. coronaria) (Jacq.) posses promising anti-TB activity against Mycobacterium tuberculosis (M. tuberculosis) H37Rv with minimum inhibitory concentrations (MICs) of 200–100 µg/mL. This study aimed to investigate the interactions of these active fractions with first-line anti-TB drugs (isoniazid, rifampicin, ethambutol and streptomycin) against M. tuberculosis H37Rv using the microdilution checkerboard method. C. citratus (stem-rhizome) n-hexane fraction exhibited synergism with all drugs except ethambutol which showed additive interaction. Synergistic was also observed when C. speciosus (stem-flower) n-hexane and T. coronaria (leaf) n-hexane fractions in combination with rifampicin. C. speciosus (stem-flower) n-hexane and T. coronaria (leaf) n-hexane exhibited additive interaction with isoniazid, ethambutol and streptomycin. Hence, these active plants are worthy of further investigations for the discovery of anti-TB drug leads.

1390. Durlobactam, a Diazabicyclooctane (DBO) β-lactamase Inhibitor (BLI), Inhibits BlaC and Peptidoglycan (PG) Transpeptidases of Mycobacterium tuberculosis (Mtb): A Novel Approach to Therapeutics for Tuberculosis (TB)?

Background Novel therapies for multidrug-resistant TB are needed and new BLIs could answer this call. Mtb encodes for BlaC, a class A β-lactamase. BlaC is inhibited by clavulanate (CLA) while the DBO avibactam (AVI) is an inefficient inhibitor (low k2/K value). Carbapenems are hydrolyzed slowly by BlaC (low kcat/Km value) making them “dual action” compounds that inhibit both BlaC and PG transpeptidases, the intended β-lactam targets. DBOs inhibit PG transpeptidases in other bacteria. To explore the therapeutic potential of new DBOs against Mtb, we compared the inhibitor activity of AVI, relebactam (REL), and durlobactam (DUR, formerly ETX2514) against BlaC and Mtb PG transpeptidases using a biochemical approach. We also investigated the ability of DUR to lower minimum inhibitory concentrations (MICs) of β-lactams against Mtb H37Rv. Methods Mass spectrometry was performed to capture acyl-enzyme complexes (AECs) of purified BlaC and PG transpeptidases (PonA1, LdtMt1, LdtMt2, LdtMt3, and LdtMt5) with β-lactams and BLIs. Steady-state enzyme kinetics were determined using nitrocefin as a substrate. MICs with amoxicillin (AMX), meropenem (MER), CLA, and DUR alone and in combination against Mtb H37Rv were assessed using a microdilution method. Results DUR alone had a MIC of 2 µg/mL with Mtb H37Rv (Table 1). BlaC formed AECs with all carbapenems and BLIs. BlaC had lower Ki app and higher k2/K with DUR than those with AVI and REL and comparable to those with CLA; however, with a period of pre-incubation, AVI fully inhibits BlaC (Table 2). The carbapenems and DUR formed the most AECs with PG transpeptidases of the β-lactams and BLIs respectively; PG transpeptidases had lower Ki app values with DUR than those with AVI (Table 3). Table 1. Minimum Inhibitory Concetrations for Mycobacterium tuberculosis H37Rv Conclusion DUR alone has some antimicrobial activity against Mtb H37Rv. The likely mechanism that underlies this activity is inhibition of BlaC and several PG transpeptidases. Inhibition of enzyme targets with DUR was more potent and efficient than AVI and REL. DUR in combination with β-lactams lowered MICs but the DUR concentration used was higher than its MIC. Our findings support the exploration of novel BLIs against BlaC and PG transpeptidases with the ultimate goal of repurposing these drugs for the treatment of TB. Disclosures Robert A. Bonomo, MD, entasis (Research Grant or Support)Merck (Grant/Research Support)NIH (Grant/Research Support)VA Merit Award (Grant/Research Support)VenatoRx (Grant/Research Support)

Syntheses and Structure–Activity Relationships of N-Phenethyl-Quinazolin-4-yl-Amines as Potent Inhibitors of Cytochrome bd Oxidase in Mycobacterium tuberculosis

The development of cytochrome bd oxidase (cyt-bd) inhibitors are needed for comprehensive termination of energy production in Mycobacterium tuberculosis (Mtb) to treat tuberculosis infections. Herein, we report on the structure-activity-relationships (SAR) of 22 new N-phenethyl-quinazolin-4-yl-amines that target cyt-bd. Our focused set of compounds was synthesized and screened against three mycobacterial strains: Mycobacterium bovis BCG, Mycobacterium tuberculosis H37Rv and the clinical isolate Mycobacterium tuberculosis N0145 with and without the cytochrome bcc:aa3 inhibitor Q203 in an ATP depletion assay. Two compounds, 12a and 19a, were more active against all three strains than the naturally derived cyt-bd inhibitor aurachin D.