Investigation on the substrate specificity and N-substitution tolerance of PseF in catalytic transformation of pseudaminic acids to CMP-Pse derivatives.

Pseudaminic acid (Pse) belongs to a class of bacterial non-2-ulosonic acids, and has been implicated in bacterial infection and immune evasion. Various Pse structures with diverse N-substitutions have been identified in pathogenic bacterial strains like Pseudomonas aeruginosa, Campylobacter jejuni, and Acinetobacter baumannii. In this study, we successfully synthesized three new Pse species, including Pse5Ac7Fo, Pse5Ac7(3RHb) and Pse7Fo5(3RHb) using chemical methods. Furthermore, we investigated the substrate specificity of cytidine 5'-monophosphate (CMP)-Pse synthetase (PseF), resulting in the production of N-modified CMP-Pse derivatives (CMP-Pses). It was found that PseF was promiscuous with the Pse substrate and could tolerate different modifications at the two nitrogen atoms. This study provides valuable insights into the incorporation of variable N-substitutions in the Pse biosynthetic pathway.

KpsS1 Mediates the Glycosylation of Pseudaminic Acid in Acinetobacter Baumannii.

Pseudaminic acid (Pse) is found in the polysaccharide structures of the cell surface of various Gram-negative pathogenic bacteria including Acinetobacter baumannii and considered as an important component of cell surface glycans including oligosaccharides and glycoproteins. However, the glycosyltransferase that is responsible for the Pse glycosylation in A. baumannii remains unknown yet. In this study, through comparative genomics analysis of Pse-positive and negative A. baumannii clinical isolates, we identified a potential glycosyltransferase, KpsS1, located right downstream of the Pse biosynthesis genetic locus. Deletion of this gene in an Pse-positive A. baumannii strain, Ab8, impaired the glycosylation of Pse to the surface CPS and proteins, while the gene knockout strain, Ab8ΔkpsS1, could still produce Pse with 2.86 folds higher amount than that of Ab8. Furthermore, impairment of Pse glycosylation affected the morphology and virulence potential of A. baumannii, suggesting the important role of this protein. This study will provide insights into the further understanding of Pse in bacterial physiology and pathogenesis.

Discovery of a Monoclonal Antibody That Targets Cell-Surface Pseudaminic Acid of Acinetobacter baumannii with Direct Bactericidal Effect.

The therapeutic effects of antibodies include neutralization of pathogens, activation of the host complement system, and facilitation of phagocytosis of pathogens. However, antibody alone has never been shown to exhibit bactericidal activity. In this study, we developed a monoclonal antibody that targets the bacterial cell surface component Pseudaminic acid (Pse). This monoclonal antibody, Pse-MAB1, exhibited direct bactericidal activity on Acinetobacter baumannii strains, even in the absence of the host complements or other immune factors, and was able to confer a protective effect against A. baumannii infections in mice. This study provides new insight into the potential of developing monoclonal antibody-based antimicrobial therapy of multidrug resistant bacterial infections, especially those which occurred among immunocompromised patients.

Identification of a KPC Variant Conferring Resistance to Ceftazidime-Avibactam from ST11 Carbapenem-Resistant Klebsiella pneumoniae Strains.

A novel Klebsiella pneumoniae carbapenemase (KPC) variant, KPC-93, was identified in two Klebsiella pneumoniae clinical isolates from a patient from China treated with ceftazidime-avibactam. KPC-93 possessed a five-amino-acids insertion (Pro-Asn-Asn-Arg-Ala) between Ambler positions 267 and 268 in KPC-2. Cloning and expression of the blaKPC-93 gene in Escherichia coli, followed by determination of minimum inhibitory concentration (MIC) values and kinetic parameters, showed that KPC-93 exhibited increased resistance to ceftazidime-avibactam, but a drastic decrease in carbapenemase activity. Our data highlight that a KPC variant conferring resistance to ceftazidime-avibactam could be easily induced by ceftazidime-avibactam treatment and that actions are required to control dissemination of these determinants. IMPORTANCE Ceftazidime-avibactam (CZA) is a novel ß-lactam/ß-lactamase inhibitor combination with activity against serine ß-lactamases, including the Ambler class A enzyme KPC. However, during recent years, there have been increasing reports of emergence of new KPC variants that could confer resistance to CZA. This has limited its clinical application. Here, we reported a new KPC variant, KPC-93, that could confer CZA resistance. KPC-93 possessed a five-amino-acids insertion (Pro-Asn-Asn-Arg-Ala) between Ambler positions 267 and 268 in KPC-2. Our findings have revealed the potential risk of blaKPC gene mutations associated with CZA exposure over a short period of time.

Unveiling the evolution routes of TEM-type extended-spectrum ß-lactamases.

The TEM-1 ß-lactamase can only cleave penicillin and the first-generation cephalosporins but it has evolved to become active against second-, third- and fourth-generation drugs. Through sequence analysis of natural TEM variants and those created by mutagenesis experiments, we described two distinct evolution routes of TEM-1 that has generated over 220 enzyme variants. One began with the Gly238Ser alteration and the other originated with the Arg164Ser substitution. Further acquisition of mutations in the background of each of these two first-step mutants led to stepwise alteration in enzyme structure and hence activity, eventually producing a wide range of enzyme variants whose substrate specificities cover cephalosporins of all generations. Dissemination of strains producing TEM-1 variants generated from these two evolution routes underlies the markedly increased prevalence of bacterial resistance to ß-lactams in the past few decades. This study provides insights into the evolution of hydrolysing enzymes, in particular ß-lactamases.

Total Synthesis of Mannopeptimycin ß via ß-Hydroxyenduracididine Ligation.

Nonribosomal peptide synthesis in bacteria has endowed cyclic peptides with fascinating structural complexity via incorporating nonproteinogenic amino acids. These bioactive cyclic peptides provide interesting structural motifs for exploring total synthesis and medicinal chemistry studies. Cyclic glycopeptide mannopeptimycins exhibit antibacterial activity against antibiotic-resistant Gram-positive pathogens and act as the lipid II binder to stop bacterial cell wall biosynthesis. Here, we report a strategy streamlining solution phase-solid phase synthesis and chemical ligation-mediated peptide cyclization for the total synthesis of mannopeptimycin ß.