Kristian Riesbeck

Background: Regulation of transcription is essential for the pathoadaptation of non-typeable Haemophilus influenzae (NTHi). We identified fatty acid biosynthesis repressor (FabR) as a previously unrecognized transcriptional regulator in NTHi through its binding to the promoter region of ompP5 and investigated its contribution to bacterial virulence and physiology. Methods: A fabR deletion mutant (ΔfabR) was generated in NTHi strain 3655. The mutant was compared with the wild-type strain in assays of host cell adherence, serum resistance, immunoglobulin G (IgG) deposition, vitronectin binding, murine nasopharyngeal colonization, protein expression, membrane fatty acid composition, and metabolic activity. Metabolic activity was also evaluated in ΔfabR mutants generated in additional NTHi strains. Results: Deletion of fabR reduced host cell adherence, increased serum sensitivity, enhanced IgG deposition, and decreased vitronectin binding compared with the wild-type strain. The ΔfabR mutant also exhibited impaired colonization of the murine nasopharynx. Proteomic analyses demonstrated altered expression of metabolic proteins, accompanied by an increased unsaturated-to-saturated fatty acid ratio. Furthermore, ΔfabR mutants from multiple NTHi strains showed increased metabolic activity, indicating a conserved role for FabR in metabolic regulation. Conclusions: FabR is a novel transcriptional regulator that may contribute to NTHi virulence. By modulating bacterial metabolism and membrane lipid composition, FabR may influence outer membrane architecture, thereby affecting host interaction, immune evasion, and colonization.

Introduction: Determining the etiology of community-acquired pneumonia (CAP) is essential for targeted treatment and optimal care. Reliable results require selection of the appropriate sampling site. Methods: Paired nasopharyngeal (NP) and oropharyngeal (OP) swabs were collected from 484 patients with CAP between September 2016 and September 2018. The samples were analyzed by PCRs for Streptococcus pneumoniae , Haemophilus influenzae , 6 atypical bacterial species, and 14 viral agents. Results: The most common pathogen was S. pneumoniae (24%), followed by H. influenzae (23%). Both were more frequently detected in the oropharynx, with 29% and 40% respectively, identified only in OP swabs. Respiratory viruses were found in 32% of the patients; of these, 65% were present in both NP and OP samples. Notably, 21% were detected only in NP samples, and 15% only in OP samples. Mycoplasma pneumoniae was identified in 6% of all patients, predominantly in OP samples (89%) compared with NP samples (70%). Cq-values were lower in NP swabs for all pathogens except M. pneumoniae , suggesting a higher microbial load in the nasopharynx. Discussion: This study highlights the importance of sampling site selection in the diagnosis and management of CAP. Nasopharyngeal sampling was more effective for detecting respiratory viruses, whereas OP samples yielded more cases of S. pneumoniae , H. influenzae and M. pneumoniae . To maximize the diagnostic yield, a combined NP/OP-sampling strategy can thus be recommended.

Background: Escalating resistance among Gram-negative pathogens limits β-lactam options. Cefepime/enmetazobactam combines a fourth-generation cephalosporin with a class A β-lactamase inhibitor. We evaluated its activity against multidrug, extensive and pandrug-resistant war-wound isolates from Ukraine. Materials and methods: We tested 215 clinical samples (2022–2023) isolated from wounded patients in Ukraine by broth microdilution. Paired comparisons of cefepime monotherapy versus cefepime/enmetazobactam was done to evaluate enmetazobactam. Whole-genome sequencing (n = 83) identified β-lactamases, sequence types and outer-membrane protein alterations. Results: Across Enterobacterales, resistance decreased from 60.5% (26/43) with cefepime to 27.9% (12/43) with the cefepime/enmetazobactam combination. In K. pneumoniae, resistance declined from 92.6% (75/81) to 74% (60/81) and in P. mirabilis, from 88.9% (8/9) to 11.1% (1/9), respectively. P. aeruginosa showed a modest change [77.8% (23/36) to 58.3% (32/36)], while A. baumannii remained non-susceptible to both cefepime and cefepime/enmetazobactam with the rates of 91.3% (42/46) to 82.6% (36/46), respectively. Genotype–phenotype correlation revealed that in K. pneumoniae, reduced activity was associated with OmpK36 loop-3 insertions, OmpK35 loss and metallo-β-lactamases, whereas selected sequence types lacking these changes were more susceptible. In A. baumannii, OXA-type carbapenemases and blaADC beta-lactamases predominated; isolates lacking type VI secretion, omp33-36 and adhesin genes (ata/bap) demonstrated relatively higher susceptibility to cefepime/enmetazobactam. Conclusions: Cefepime/enmetazobactam improved susceptibility relative to cefepime, most notably in Enterobacterales, but activity was constrained by permeability defects and non-target β-lactamases. Combination therapy with agents that does not get hydrolysed by MBLs and integrated β-lactamase detection with practical permeability indicators may optimize the clinical use of this combination in high-resistance settings.

Acquisition of complement regulators is a virulence strategy used by nontypeable Haemophilus influenzae (NTHi) to evade complement-mediated killing by the host. The major outer membrane protein of NTHi, P5, binds C4b-binding protein and factor H to promote bacterial serum resistance. We show that P5 also binds vitronectin, which inhibits the formation of the membrane attack complex at the terminal stage of the complement pathway. Heterologous surface expression of P5 variants from NTHi strains 3655, KR271, KR317, and P652 promoted vitronectin binding to the P5-expressing Escherichia coli. In contrast, deletion of P5 from the NTHi strains reduced vitronectin binding. Vitronectin acquisition conferred serum resistance to P5-expressing E. coli, but not to NTHi ΔompP5 mutants. Using site-directed mutagenesis, extracellular loop 2 of the P5 variants was identified as the binding site for vitronectin. In conclusion, our findings highlight P5 as a receptor for vitronectin that promotes NTHi serum resistance.

Messenger RNA (mRNA) vaccines based on lipid nanoparticles (LNPs) are stabilized with cholesterol, which is thought to be a critical LNP component because it is essential for membrane integrity and endosomal escape. Here, it is shown that cholesterol in LNPs can be replaced with an immunopotentiating lipid, i.e., a synthetic analogue of the C‐type lectin receptor agonist monomycoloyl glycerol (MMG‐1), without compromising physicochemical properties, in vivo transfection efficiency, and immunogenicity of the mRNA‐loaded LNPs (mRNA‐LNPs). Replacement of cholesterol with MMG‐1 results in LNPs that mediate intracellular delivery of mRNA, which is translated into high levels of protein in vivo. Replacement of cholesterol with MMG‐1 in LNPs improves the transfection efficiency in T cells, B cells, and macrophages in the spleen and lymph nodes of mice. In mice, MMG‐1‐based LNPs loaded with mRNA encoding the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) induce S‐specific CD8 ⁺ T‐cell responses and virus‐neutralizing antibody responses, which are on par with the responses induced by cholesterol‐based LNPs. Upon vaccination and a subsequent SARS‐CoV‐2 challenge of Syrian golden hamsters, replacement of cholesterol with MMG‐1 in mRNA‐LNPs enhances S‐specific immunoglobulin G titers and reduces the SARS‐CoV‐2 load in the nasal cavity. These findings provide insights for improved design of LNPs for mRNA vaccine delivery.

The disruption of microtubule dynamics serves as a pivotal strategy for eliminating tumor cells, despite its accompanying toxicities affecting non‐tumor cells. This study investigates the potential of selectively targeting γ‐tubulin1 (TUBG1) as a therapeutic strategy in cancer treatment. By elucidating the TUBG1–E2F1–retinoblastoma protein (RB1) network, we introduce a novel compound, 4‐(6‐((3‐Methoxyphenyl)amino)pyrimidin‐4‐yl)‐N,N‐dimethylbenzenamine, (L12). L12 treatment enhanced RB1 expression and selectively targeted cells with impaired RB1 signaling, while reduced E2F1 expression attenuated its cytotoxicity. Furthermore, L12‐mediated cytotoxicity depends on an E2F1‐mediated upregulation of procaspase 3 expression, highlighting the role of E2F1 in the apoptotic response. Unlike traditional tubulin‐targeting agents, L12's specificity for tumor cells lies in its inhibitory effects on TUBG1, without affecting the second human isoform of TUBGs, TUBG2. Despite its interaction with specific kinases, the concentrations required for antitumor effects are 100‐fold lower than those influencing kinase activities. Subsequent investigations underscore L12's reduced neuronal axonal toxicity compared to vincristine. Lastly, L12 demonstrates promising results in inhibiting tumor growth in xenografted small cell lung cancer models, demonstrating potential specificity toward tumor cells while minimizing adverse effects on healthy tissues. This research emphasizes the potential of TUBG1 inhibitors as a promising advancement in personalized chemotherapy approaches and their potential as a groundbreaking treatment for various cancers.