Macrolide overuse and resulting resistance crippled the applicability of one of the most important and successful classes of antibiotics. The rising incidence of invasive infections caused by β-hemolytic group A and group B streptococci have been paralleled with marked increases in erythromycin and clindamycin resistance. As a result, resistant invasive group A and group B Streptococcus infections have been classified by the CDC as a concerning level threat since 2019. Acquisition of erythromycin resistance methylase (erm) genes is a major contributor of the MLSB [macrolide (i.e., erythromycin), lincosamide (i.e., clindamycin), and streptogramin B] resistance phenotype in US isolates. The erm-encoded enzymes utilize the methyl-group from S-adenosyl-L-methionine (SAM) to methylate the adenosine A2058 residue of bacterial ribosomal RNA, which serves as the overlapping rRNA target site for the three MLSB classes of antibiotics. The major mechanism that regulates Erm methyltransferase production is translational attenuation in the 5' regulatory region upstream of the erm coding sequence. The 5' regulatory region of erm includes at least one short leader peptide ermL, with the leader peptide ribosome binding site (SD1), non-translational stem-loop structures, and the erm start codon with its ribosome binding site (SD2). Binding of a macrolide antibiotic to the ribosome causes stalling during translation of the ermL peptide, which disrupts and alters the formation of inhibitory mRNA hairpins in the erm 5' regulatory region, thereby releasing the erm SD2-site from the posterior stem-loop hairpin, thus allowing Erm protein translation. The streptococcal ErmA, ErmB, and ErmT proteins have conserved structures with primary sequence conservation of residues involved in SAM- and rRNA-binding activity, as well as a high-level of structural conservation. However, the observed variation in the erm 5' regulatory region sequences, transcript expression, and ribosome methylation levels associated with these genes and proteins underscore the regulatory complexity of Erm expression. This review aims to present insights into mechanisms of Erm-mediated resistance, trends in streptococcal epidemiology and treatment, and progress in the development of improved MLSB drugs that effectively block erm-mediated resistance.

Our understanding of how depolymerase sequence and structure determine substrate specificity is fragmentary due to the limited number of experimentally characterized enzymes. Here we show DepoCatalog - an experimentally validated collection of 129 recombinantly prepared Klebsiella phage depolymerases (90 enzymes produced in this study and 39 homologs from the literature), with specificity spanning 75 KL-types. Enzymes originated from podo-, sipho-, myo-, jumbo phages, and prophages. Using activity profiling, structural modeling, and domain dissection, we propose a five‑class framework that captures the architectural and functional diversity of these enzymes. DepoCatalog uncovers cross-reactivity and taxa‑specific enzymes. Structural comparisons indicate that specificity switching or extension is associated with modifications to the C‑terminal domain. We further hypothesize that podoviruses encoding up to two RBPs show greater receptor adaptability than jumbo phages with multiple specialized RBPs. Finally, we develop a publicly accessible, DepoCat dataset ( https://depocat.uwr.edu.pl ) for specificity, structural classification and comparison of newly identified depolymerases.

HtpGMtb of Mycobacterium tuberculosis is an ATP-dependent heat shock protein that assists the correct folding of nascent and stress-accumulated misfolded proteins, in concert with other chaperones. Besides playing a role in stress response, it is able to elicit an immune response against M. tuberculosis infection by activating Dendritic Cells in a Toll Like Receptor 4-mediated manner. However, we lack a full understanding of the molecular determinants of HtpGMtb catalytic activity and Toll Like Receptor 4 activation, due to the lack of structural and biophysical data. Here, we report the first crystal structure of HtpGMtb, in complex with the non-hydrolysable form of ATP. The crystal structure reveals that the HtpGMtb dimer adopts a conformationally silent structure, that precludes the dimerisation of the chaperone catalytic domains needed for ATP hydrolysis. Also, binding studies show that HtpGMtb directly interacts with Toll Like Receptor 4 with a nanomolar affinity, and that this interaction allows HtpGMtb dimer to engage two host receptor molecules. This finding suggests that activation of Toll Like Receptor 4 by HtpGMtb is due to its ability to induce the dimerisation of the host receptor, an essential step for initiating the entire signaling cascade.

The global rise of multidrug-resistant bacteria is a major health threat, with Klebsiella pneumoniae identified by the WHO as a critical priority pathogen. Phage-derived depolymerases have emerged as promising countermeasures because they can degrade the capsular polysaccharide (CPS) that shields bacteria from the immune system, thereby increasing their susceptibility to antibiotics and host defences. However, the limited understanding of how they recognise specific CPS structures remains a major obstacle to developing effective depolymerase-based therapeutics. In this study, we provide a comprehensive NMR and mutational analysis to characterise the mechanism of action of a miniaturised depolymerase, here mKP34gp57, targeting the clinically relevant K63 CPS. We show by NMR that mKP34gp57 hydrolyses CPS with high efficiency through an endoglycosidase-retaining mechanism. During substrate recognition, our data demonstrate that the enzyme interacts predominantly with the galactose and fucose moieties, which serve as the critical recognition features and thus the principal determinants of CPS specificity. Computational studies provide structural clues for the roles of the catalytic residues E266/E300 and D151. Finally, we prove that the hexasaccharide produced upon CPS hydrolysis stimulates dendritic cell maturation and T-helper-driven lymphocyte proliferation. Identifying the binding determinants that govern CPS recognition by mKP34gp57, and using this information to generate immunogenic fragments, deepens our understanding of how minidepolymerases can be rationally engineered to achieve tailored serotype specificity, and improved therapeutic and diagnostic potential.

After the global eradication of smallpox has occurred, Monkeypox virus (MPXV) is nowadays the most significant pathogen affecting humans among orthopoxviruses. The recent growing number of cases worldwide is drawing researchers' attention, prompting for the discovery of new antivirals with optimized synthetic protocols and enhanced efficacy. To date tecovirimat, targeting the membrane-anchored phospholipase p37, is the only drug specifically approved for the treatment of MPXV. In an effort to develop inhibitors with more accessible synthetic protocols and broaden the poorly explored structure-activity relationship (SAR) studies, a new library of tecovirimat analogues has been designed and synthesized. The resulting compounds have been tested in phenotypic assays to evaluate their antiviral inhibitory properties. Spirovirimat (7) was identified as a potent lead compound within this series. Further experiments highlighted that 7 completely abolished extracellular virus production, and in silico studies suggested that 7 and tecovirimat target the same protein.

DEAD-box protein (DDX) 5 plays important roles in multiple aspects of cellular processes that require modulation of the RNA structure. Alongside the canonical role in RNA metabolism, numerous studies have demonstrated that DDX5 influences viral infections by directly interacting with viral proteins. However, the precise functional role of DDX5 during viral infection remains largely unclear. Here, we explore the previously undiscovered ability of DDX5 to interact and synergize with the Nsp13 helicase of SARS-CoV-2. We show that DDX5 exhibits a nanomolar binding affinity to Nsp13. Also, by dissecting DDX5 in its individual domains, we show that the Nsp13-DDX5 interaction is mediated by the RecA1 domain of DDX5. Importantly, we show that DDX5 and Nsp13 synergize in unwinding double-stranded RNA. Consistent with its ability to bind Nsp13, the RecA1 domain of DDX5 acts as a weak inhibitor of the synergic action of the two helicases in the RNA unwinding process. Modeling of the DDX5-Nsp13 complex provides a plausible explanation for the synergic action of the two helicases, in a mechanism that is likely instrumental in the early stage of infection, when the concentration of Nsp13 is still low.

Tuberculosis remains a critical global health challenge, which underscores the need for new therapeutic targets. A potential drug target is the rhodanese-like thiosulfate sulfurtransferase SseA, which plays a role in macrophage infection by Mycobacterium tuberculosis (Mtb) and its resistance to oxidative stress. In our research, we identified a protein (Rv3284), herein referred to as SufEMtb, that interacts with SseA and modulates its activity. Sequence analysis and molecular modeling revealed that SufEMtb enhances SseA enzymatic function by binding to its non-catalytic N-terminal domain and favoring an activating conformational change in a regulatory loop of SseA. This interaction appears crucial for effective enzyme activity and the maintenance of redox homeostasis in Mtb, making the SseA-SufEMtb complex a potential target for new therapies.

HtpG of Mycobacterium tuberculosis (HtpGMtb) is an ATP-dependent chaperone that assists the correct folding of nascent and stress-accumulated misfolded proteins, in concert with other chaperones. Beside playing a role in stress response, it is able to elicit an immune response against M. tuberculosis infection by activating Dendritic Cells in a TLR4-mediated manner. However, we lack a full understanding of the molecular determinants of HtpGMtb catalytic activity and TLR4 activation, due to the lack of structural and biophysical data. Here, we report the first crystal structure of HtpGMtb, in complex with the non-hydrolysable form of ATP, AMPPNP. The crystal structure reveals that the HtpGMtb dimer adopts a conformationally silent structure, that precludes the dimerisation of the chaperone catalytic domains needed for ATP hydrolysis. Also, binding studies show that HtpGMtb directly interacts with TLR4 with a nanomolar affinity, and that this interaction allows HtpGMtb dimer to engage two TLR4 molecules. This finding suggests that activation of TLR4 by HtpGMtb is due to its ability to induce intra-cellular receptor dimerisation in an LPS-like mode.

Enterococcus faecium have high rates of antibiotic resistances, with vancomycin-resistant E. faecium acknowledged as the most important in the clinical setting and declared by WHO to be a threat to humankind, for which rapid actions are needed. PpiC is a membrane-bound lipoprotein of E. faecium endowed with both a peptidyl-prolyl isomerase and a foldase activity, and plays a key role in assisting the folding of many secreted enterococcal proteins. It is located at the membrane-wall interface, therefore easily accessible to inhibitors and to the immune system and an ideal target for drug and vaccine development. Despite their potential, enterococcal peptidyl-prolyl isomerases have been understudied. We previously identified PpiC as an important cross-protective vaccine antigen. To gain a better understanding of the PpiC biological role in E. faecium survival, we determined the crystal structure of PpiC and investigated its biophysical properties. Consistent with PpiC's folding activity, the biological assembly of PpiC is a bowl-shaped structure containing two parvulin-type peptidyl-prolyl cis/trans isomerase domains. We also dissected the role of N- and C-terminal regions of the molecule in its dimerisation, an event which is predicted to play an important role in the folding of client proteins. Our data point to a functional cross-talk between the foldase and peptidyl-prolyl isomerase activities of PpiC, through the protein-swapping involved in dimerisation. Also, our work provides key structural data for the design of antimicrobials and cross-protective vaccine antigens against nosocomial infections.

IntroductionGroup B Streptococcus (GBS) is a leading etiological agent of neonatal sepsis and meningitis, as well as invasive infections that predominately affect adults over 65 and people with comorbidities. The Centers for Disease Control and Prevention’s (CDC) nationwide surveillance has identified rising clindamycin and erythromycin resistance as a concerning level threat for invasive GBS infections. West Virginia (WV) is a rural state outside the CDC catchment area, therefore, we investigated 65 WV-GBS isolates identified in clinical specimens from various sources for serotype distribution, as well as the spectrum and genetic markers of erythromycin and clindamycin resistance.ResultsGBS isolates of serotypes II (27.7%), Ib (23.1%), V (18.5%), Ia (13.8%), III (10.8%), and IV (6.2%), were identified. The mef(A) gene encoding macrolide resistance (M phenotype) was detected in 15.4% isolates. The erm(A) and erm(B) genes that are responsible for the combined erythromycin and clindamycin resistance, which characterizes an MLSB [macrolide, lincosamide, and streptogramin B] resistance phenotype, were detected in 67.7% of WV-GBS isolates; also, 81.4% of those displayed constitutive (cMLSB) and 18.6% inducible (iMLSB) resistance to clindamycin by erythromycin using the D-test. A cluster of mutations within the regulatory region of erm(A) were identified in association with a cMLSB sub-phenotype, whereas most of the erm(B) promoters sequenced from isolates with a cMLSB background lacked analogous sequence polymorphisms. Further, higher erythromycin MIC values were associated with the erm(B) determinant compared with erm(A), while structural models of the GBS-ErmA and GBS-ErmB enzymes show conservation in both SAM- and rRNA-binding sites.SignificanceOur data demonstrate that the 80.0% rate of erythromycin and 70.8% of clindamycin resistance in WV is higher than the national average of 61.7 and 52.5%, respectively, while being widespread across a variety of clinical specimens (urine, throat, respiratory tract, blood, foot ulcers, perisplenic fluid, various wounds, and the rectovaginal area). Providers should be aware of the current threat of antibiotic resistance, especially in “primary care deserts” existing in rural areas such as WV.

Background: Klebsiella pneumoniae is one of the most critical Gram-negative bacteria according to the World Health Organization (WHO). Due to the ability of this bacterium to evade antibiotics, phage therapy is becoming a promising tool. However, the use of isolated proteins rather than entire phages could reduce several risks associated with phage replication. Thus, understanding the protein composition and structural organization of bacteriophages is crucial for unlocking their biology and holds great potential for medicine and biotechnology. Methods: In this study, artificial intelligence with AlphaFold 3.0 (AF3) and bioinformatic analysis were used to model the hitherto unknown structure of the Klebsiella phage KP32 (KP32), a complex and selective phage that targets K. pneumoniae strains with the K3 and K21/KL163 capsular serotypes. Results: By combining AF3 with sequence and structure analysis, we reconstructed the entire phage KP32. This complex phage is composed of over 500 protein chains, of which 415 compose its capsid and 104 its core-portal-tail complex, a platform that allows the phage to adhere to K. pneumoniae, hydrolyze its capsular sugars and finally inject its genetic code into the bacterium. Conclusions: Phage therapy is a potentially promising tool for controlling antimicrobial resistance (AMR). However, one limitation arises from the limited knowledge of their nature and mechanisms of action, as only a few phages have been structurally characterized. The reconstruction of entire phages is currently a viable strategy for elucidating their mechanistic properties, knowledge that will enhance their potential applications as therapeutic alternatives.

OBJECTIVE:Klebsiella pneumoniae Przondovirus KP32 presents a complex capsular degradation machinery comprised of two serotype-specific depolymerases, KP32gp38 and KP32gp37. METHODS:In this work, we performed capsular polysaccharide (CPS) degradation assays combined with mass spectrometry approaches to identify the reaction product of K21 serotype CPS degradation by KP32gp38. We determined the crystal structure of the KP32gp38 depolymerase in complex with the identified degradation product, a pyruvated pentasaccharide, called K21-pyr5. RESULTS:The structure showed that K21-pyr5 binds to the inter-chain catalytic site, allowing the identification of important residues for CPS recognition. Importantly, we observed that the production of K21-pyr5 through CPS degradation by KP32gp38 is able to induce the maturation and differentiation of monocyte-derived dendritic cells, which, in turn, induce lymphocyte proliferation and Th polarization. By employing a T7 phage of Escherichia coli analogy, we were able to provide insights into the portal assembly of the Przondovirus K32. Our modeling studies suggest that the KP32 portal, attached to its icosahedral capsid shell, carries 12 depolymerase molecules on a single virion, arranged in 6 branches; in each branch, KP32gp38 depolymerase adheres to KP32gp37, which is directly connected to the phage portal. CONCLUSIONS:Overall, our results suggest that depolymerases act as anti-virulent agents, not only by depleting the bacteria of their CPS but also by producing immunostimulatory CPS degradation products. This indicates the use of CPS degradation products by depolymerases as potential antigens in K. pneumoniae vaccination strategies.

A validated catalog of 105 recombinant depolymerases from Klebsiella phages covers 58 KL-types. 46 novel enzymes from prophages, jumbo phages, and common phages are linked to any known enzymatic activity against 14 classical serotypes and 12 genome-defined KL-types. Using activity-based profiling, structure prediction, and domain dissection, we developed a function-guided classification and a five-class structural catalog. This framework reveals highly specific enzymes active against up to three capsule types. K47 CPS was degraded by three diverse protein groups. Structurally similar depolymerases degrading particular CPS were found in distinct phage taxa, with highly conserved enzymes in Drulisvirus specific to K1-, K2-types. The exclusive depolymerases were found in siphoviruses targeting K2 and K62 serotypes. A case study of five structurally similar enzymes degrading KL22/KL37/KL111 and KL25/KL119 capsules suggested specificity switching via amino acid changes or C-domain modification. Klebsiella phage depolymerases catalog sheds light on their diversity, evolution, and potential application. ### Competing Interest Statement The authors have declared no competing interest. Narodowe Centrum Nauki, UMO-2017/26/M/NZ1/00233, UMO-2022/47/I/NZ1/01450, UMO-2020/38/E/NZ8/00432, UMO-2022/04/Y/NZ6/00123 Polish National Agency for Academic Exchange Agence Nationale de la Recherche, https://ror.org/00rbzpz17, ANR-20-AMRB-0004-01, ANR-22-AAMR-0006-06 Research Foundation–Flanders, 1240021N, 1251224N The Federal Ministry of Education and Research, 1KI2302A

Tuberculosis (TB) remains a critical global health challenge, with Mycobacterium tuberculosis (Mtb) causing 10.8 million cases and 1.25 million deaths in 2023. Key issues include the dormant state of Mtb, resistant to drugs and immune responses, and the emergence of multi-drug-resistant strains. This underscores the need for new therapeutic targets and deeper research into Mtb pathogenesis and immunology. A potential drug target is the enzyme thiosulfate-sulfurtransferase SseA, which plays a role in macrophage infection by Mtb and its resistance to oxidative stress. SseA belongs to the rhodanese-like enzyme family, which catalyzes sulfur transfer reactions essential for Mtb survival. In our research, we identified a new protein (Rv3284), hereinafter referred to as SufEMtb due to its high homology with E. coli SufE, that interacts with SseA and modulates its activity. Sequence analysis and AI molecular modelling revealed detailed insights into their interaction that can contribute to the modulation of SseA activity. This research provides a mechanistic explanation to the need of a partner for SseA activation. Indeed, we propose that SufEMtb enhances SseA enzymatic function by binding to its non-catalytic N-terminal domain and bringing the active sites of the two proteins in close proximity, thus preparing for the activation-enhancing conformational change in a regulatory loop of SseA. This interaction is crucial for the effective enzyme activity and the maintenance of redox homeostasis in Mtb, making the SseA-SufEMtb protein complex a potential target for new TB therapies. ### Competing Interest Statement The authors have declared no competing interest.

ESKAPE pathogens are responsible for complicated nosocomial infections worldwide and are often resistant to commonly used antibiotics in clinical settings. Among ESKAPE, vancomycin-resistant Enterococcus faecium (VREfm) and methicillin-resistant Staphylococcus aureus (MRSA) are two important Gram-positive pathogens for which non-antibiotic alternatives are urgently needed. We previously showed that the lipoprotein AdcA of E. faecium elicits opsonic and protective antibodies against E. faecium and E. faecalis. Prompted by our observation, reported here, that AdcA also elicits opsonic antibodies against MRSA and other clinically relevant Gram-positive pathogens, we identified the dominant epitope responsible for AdcA cross-reactive activity and designed a hyper-thermostable and multi-presenting antigen, Sc(EH)3. We demonstrate that antibodies raised against Sc(EH)3 mediate opsonic killing of a wide-spectrum of Gram-positive pathogens, including VREfm and MRSA, and confer protection both in passive and active immunisation models. Our data indicate that Sc(EH)3 is a promising antigen for the development of vaccines against different Gram-positive pathogens.

BACKGROUND:Enterococcus faecium and Staphylococcus aureus are the Gram-positive pathogens of the ESKAPE group, known to represent a great threat to human health due to their high virulence and multiple resistances to antibiotics. Combined, enterococci and S. aureus account for 26% of healthcare-associated infections and are the most common organisms responsible for blood stream infections. We previously showed that the peptidyl-prolyl cis/trans isomerase (PPIase) PpiC of E. faecium elicits the production of specific, opsonic, and protective antibodies that are effective against several strains of E. faecium and E. faecalis. Due to the ubiquitous characteristics of PPIases and their essential function within Gram-positive cells, we hypothesized a potential cross-reactive effect of anti-PpiC antibodies. RESULTS:Opsonophagocytic assays combined with bioinformatics led to the identification of the foldase protein PrsA as a new potential vaccine antigen in S. aureus. We show that PrsA is a stable dimeric protein able to elicit opsonic antibodies against the S. aureus strain MW2, as well as cross-binding and cross-opsonic in several S. aureus, E. faecium and E. faecalis strains. CONCLUSIONS:Given the multiple antibiotic resistances S. aureus and enterococci present, finding preventive strategies is essential to fight those two nosocomial pathogens. The study shows the potential of PrsA as an antigen to use in vaccine formulation against the two dangerous Gram-positive ESKAPE bacteria. Our findings support the idea that PPIases should be further investigated as vaccine targets in the frame of pan-vaccinomics strategy.