Teichoic acid (TA), a crucial cell wall constituent of the pathobiont Streptococcus pneumoniae, is bound to peptidoglycan (wall teichoic acid, WTA) or to membrane glycolipids (lipoteichoic acid, LTA). Both TA polymers share a common precursor synthesis pathway, but differ in the final transfer of the TA chain to either peptidoglycan or a glycolipid. Here, we show that LTA exhibits a different linkage conformation compared to WTA, and identify TacL (previously known as RafX) as a putative lipoteichoic acid ligase required for LTA assembly. Pneumococcal mutants deficient in TacL lack LTA and show attenuated virulence in mouse models of acute pneumonia and systemic infections, although they grow normally in culture. Hence, LTA is important for S. pneumoniae to establish systemic infections, and TacL represents a potential target for antimicrobial drug development.

Tuberculosis is a bacterial infectious disease that is mainly transmitted from human to human via infectious aerosols. Currently, tuberculosis is the leading cause of death by an infectious disease worldwide. In the past decade, the number of patients affected by tuberculosis has increased by ∼20 percent and the emergence of drug-resistant strains of Mycobacterium tuberculosis challenges the goal of elimination of tuberculosis in the near future. For the last 50 years, management of patients with tuberculosis has followed a standardized management approach. This standardization neglects the variation in human susceptibility to infection, immune response, the pharmacokinetics of drugs, and the individual duration of treatment needed to achieve relapse-free cure. Here we propose a package of precision medicine-guided therapies that has the prospect to drive clinical management decisions, based on both host immunity and M. tuberculosis strains genetics. Recently, important scientific discoveries and technological advances have been achieved that provide a perspective for individualized rather than standardized management of patients with tuberculosis. For the individual selection of best medicines and host-directed therapies, personalized drug dosing, and treatment durations, physicians treating patients with tuberculosis will be able to rely on these advances in systems biology and to apply them at the bedside.

Adequate perception of immunologically important pathogen-associated molecular patterns like lipopolysaccharide and bacterial lipoproteins is essential for efficient innate and adaptive immune responses. In the context of Gram-negative infection, bactericidal/permeability-increasing protein (BPI) neutralizes endotoxic activity of lipopolysaccharides, and thus prohibits hyperactivation. So far, no immunological function of BPI has been described in Gram-positive infections. Here, we show a significant elevation of BPI in Gram-positive meningitis and, surprisingly, a positive correlation between BPI and pro-inflammatory markers like TNFα. To clarify the underlying mechanisms, we identify BPI ligands of Gram-positive origin, specifically bacterial lipopeptides and lipoteichoic acids, and determine essential structural motifs for this interaction. Importantly, the interaction of BPI with these newly defined ligands significantly enhances the immune response in peripheral blood mononuclear cells (PBMCs) mediated by Gram-positive bacteria, and thereby ensures their sensitive perception. In conclusion, we define BPI as an immune enhancing pattern recognition molecule in Gram-positive infections.

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The Mycobacterium tuberculosis (Mtb)-harboring granuloma with a necrotic center surrounded by a fibrous capsule is the hallmark of tuberculosis (TB). For a successful treatment, antibiotics need to penetrate these complex structures to reach their bacterial targets. Hence, animal models reflecting the pulmonary pathology of TB patients are of particular importance to improve the pre-clinical validation of novel drug candidates. Mtb-infected interleukin-13 overexpressing (IL-13 tg ) mice develop a TB pathology very similar to patients and, in contrast to other mouse models, also share pathogenetic mechanisms. Accordingly, IL-13 tg animals represent an ideal model for analyzing the penetration of novel anti-TB drugs into various compartments of necrotic granulomas by matrix-assisted-laser-desorption/ionization-mass spectrometry imaging (MALDI MS imaging). In the present study, we evaluated the suitability of BALB/c IL-13 tg mice for determining the antibiotic distribution within necrotizing lesions. To this end, we established a workflow based on the inactivation of Mtb by gamma irradiation while preserving lung tissue integrity and drug distribution, which is essential for correlating drug penetration with lesion pathology. MALDI MS imaging analysis of clofazimine, pyrazinamide and rifampicin revealed a drug-specific distribution within different lesion types including cellular granulomas, developing in BALB/c wild-type mice, and necrotic granulomas of BALB/c IL-13 tg animals, emphasizing the necessity of pre-clinical models reflecting human pathology. Most importantly, our study demonstrates that BALB/c IL-13 tg mice recapitulate the penetration of antibiotics into human lesions. Therefore, our workflow in combination with the IL-13 tg mouse model provides an improved and accelerated evaluation of novel anti-TB drugs and new regimens in the pre-clinical stage.

The bacterial lung pathogen has a unique nutritional requirement for exogenous choline and attaches phosphorylcholine (-Cho) residues to the GalNAc moieties of its teichoic acids (TAs) in its cell wall. Two phosphorylcholine transferases, LicD1 and LicD2, mediate the attachment of -Cho to the O-6 positions of the two GalNAc residues present in each repeating unit of pneumococcal TAs (pnTAs), of which only LicD1 has been determined to be essential. At the molecular level, the specificity of the -Cho attachment to pnTAs by LicD1 and LicD2 remains still elusive. Here, using detailed structural analyses of pnTAs from a LicD2-deficient strain, we confirmed the specificity in the attachment of-Cho residues to pnTA. LicD1 solely transfers -Cho to α-d-GalNAc moieties, whereas LicD2 attaches -Cho to β-d-GalNAc. Further, we investigated the role of the pneumococcal phosphorylcholine esterase (Pce) in the modification of the -Cho substitution pattern of pnTAs. To clarify the specificity of Pce-mediated-Cho hydrolysis, we evaluated different concentrations and pH conditions for the treatment of pneumococcal lipoteichoic acid with purified Pce. We show that Pce can hydrolyze both -Cho residues of the terminal repeat of the pnTA chain and almost all-Cho residues bound to β-d-GalNAc However, hydrolysis was restricted to the terminal repeat. In summary, our findings indicate that LicD1 and LicD2 specifically transfer -Cho to α-d-GalNAc and β-d-GalNAc moieties, respectively, and that Pce removes distinct -Cho substituents from pnTAs.

Nontuberculous mycobacterial infections rapidly emerge and demand potent medications to cope with resistance. In this context, targeted loco‐regional delivery of aerosol medicines to the lungs is an advantage. However, sufficient antibiotic delivery requires engineered aerosols for optimized deposition. Here, the effect of bedaquiline‐encapsulating fucosylated versus nonfucosylated liposomes on cellular uptake and delivery is investigated. Notably, this comparison includes critical parameters for pulmonary delivery, i.e., aerosol deposition and the noncellular barriers of pulmonary surfactant (PS) and mucus. Targeting increases liposomal uptake into THP‐1 cells as well as peripheral blood monocyte‐ and lung‐tissue derived macrophages. Aerosol deposition in the presence of PS, however, masks the effect of active targeting. PS alters antibiotic release that depends on the drug's hydrophobicity, while mucus reduces the mobility of nontargeted more than fucosylated liposomes. Dry‐powder microparticles of spray‐dried bedaquiline‐loaded liposomes display a high fine particle fraction of >70%, as well as preserved liposomal integrity and targeting function. The antibiotic effect is maintained when deposited as powder aerosol on cultured Mycobacterium abscessus. When treating M. abscessus infected THP‐1 cells, the fucosylated variant enabled enhanced bacterial killing, thus opening up a clear perspective for the improved treatment of nontuberculous mycobacterial infections.