Towards integrated cross-sectoral surveillance of pathogens and antimicrobial resistance: Needs, approaches, and considerations for linking surveillance to action.

Pathogenic and antimicrobial-resistant (AMR) microorganisms are continually transmitted between human, animal, and environmental reservoirs, contributing to the high burden of infectious disease and driving the growing global AMR crisis. The sheer diversity of pathogens, AMR mechanisms, and transmission pathways connecting these reservoirs create the need for comprehensive cross-sectoral surveillance to effectively monitor risks. Current approaches are often siloed by discipline and sector, focusing independently on parts of the whole. Here we advocate that integrated surveillance approaches, developed through transdisciplinary cross-sector collaboration, are key to addressing the dual crises of infectious diseases and AMR. We first review the areas of need, challenges, and benefits of cross-sectoral surveillance, then summarise and evaluate the major detection methods already available to achieve this (culture, quantitative PCR, and metagenomic sequencing). Finally, we outline how cross-sectoral surveillance initiatives can be fostered at multiple scales of action, and present key considerations for implementation and the development of effective systems to manage and integrate this information for the benefit of multiple sectors. While methods and technologies are increasingly available and affordable for comprehensive pathogen and AMR surveillance across different reservoirs, it is imperative that systems are strengthened to effectively manage and integrate this information.

Genetic variation in individuals from a population of the minimalist bacteriophage Merri-merri-uth nyilam marra-natj driving evolution of the virus.

In a survey of a waterway on Wurundjeri land, two sub-populations of the bacteriophage Merri-merri-uth nyilam marra-natj (phage MMNM) were isolated on a permissive host, Klebsiella B5055 of capsule-type K2, but were distinguished by minor phenotypic differences. The variant phage MMNM(Ala134) showed an inhibited activity against Klebsiella AJ174-2, and this was used as a basis to select for further variation through experimental evolution. Over the course of an evolution experiment, 20 phages that evolved distinct phenotypes in terms of the morphologies of plaques formed when they infected host Klebsiella were subject to whole-genome sequencing. The evolved phages had mutations in a small set of proteins that contribute to the baseplate portion of the phage virion. Phages MMNM and MMNM(Ala134) are minimalist phages, with baseplates formed from only five predicted subunits, akin to other minimalist phages Pam3 and XM1. The homology between all three minimalist phages provided a structural framework to interpret the two classes of mutations derived through evolution in the presence of the semi-permissive host: those that affect the interfacial surfaces between baseplate subunits, and those in a base-plate associated tail-fiber. This study evidences that multiple small mutations can be fixed into a sub-population of phage to provide a basis for phenotypic variation that we suggest could ultimately provide for a shift of virus properties, as an alternative evolutionary scenario to the major genetic events that result in more well-studied evolutionary mechanism of phage mosaicism. IMPORTANCE: Bacteriophages (phages) are viruses that prey on bacteria. This study sampled natural phage populations to test the hypothesis that untapped genetic variation within a population can be the basis for the selection of phages to diversify their host-range. Sampling of a freshwater site revealed two populations of the phage Merri-merri-uth nyilam marra-natj (phage MMNM), differing by a variant residue (Val134Ala) in the baseplate protein MMNM_26. This sequence variation modulated bacterial killing in plaques, and further evolution of the phages on a semi-permissive bacterial host led to a new generation of phages with more diverse phenotypes in killing the bacterium Klebsiella pneumoniae.

A new model of endotracheal tube biofilm identifies combinations of matrix-degrading enzymes and antimicrobials able to eradicate biofilms of pathogens that cause ventilator-associated pneumonia.

Ventilator-associated pneumonia is defined as pneumonia that develops in a patient who has been on mechanical ventilation for more than 48 hours through an endotracheal tube. It is caused by biofilm formation on the indwelling tube, which introduces pathogenic microbes such as Pseudomonas aeruginosa, Klebsiella pneumoniae and Candida albicans into the patient's lower airways. Currently, there is a lack of accurate in vitro models of ventilator-associated pneumonia development. This greatly limits our understanding of how the in-host environment alters pathogen physiology and the efficacy of ventilator-associated pneumonia prevention or treatment strategies. Here, we showcase a reproducible model that simulates the biofilm formation of these pathogens in a host-mimicking environment and demonstrate that the biofilm matrix produced differs from that observed in standard laboratory growth medium. In our model, pathogens are grown on endotracheal tube segments in the presence of a novel synthetic ventilated airway mucus medium that simulates the in-host environment. Matrix-degrading enzymes and cryo-scanning electron microscopy were employed to characterize the system in terms of biofilm matrix composition and structure, as compared to standard laboratory growth medium. As seen in patients, the biofilms of ventilator-associated pneumonia pathogens in our model either required very high concentrations of antimicrobials for eradication or could not be eradicated. However, combining matrix-degrading enzymes with antimicrobials greatly improved the biofilm eradication of all pathogens. Our in vitro endotracheal tube model informs on fundamental microbiology in the ventilator-associated pneumonia context and has broad applicability as a screening platform for antibiofilm measures including the use of matrix-degrading enzymes as antimicrobial adjuvants.

Arginine catabolism is essential to polymyxin dependence in Acinetobacter baumannii.

Polymyxins are often the only effective antibiotics against the "Critical" pathogen Acinetobacter baumannii. Worryingly, highly polymyxin-resistant A. baumannii displaying dependence on polymyxins has emerged in the clinic, leading to diagnosis and treatment failures. Here, we report that arginine metabolism is essential for polymyxin-dependent A. baumannii. Specifically, the arginine degradation pathway was significantly altered in polymyxin-dependent strains compared to wild-type strains, with critical metabolites (e.g., L-arginine and L-glutamate) severely depleted and expression of the astABCDE operon significantly increased. Supplementation of arginine increased bacterial metabolic activity and suppressed polymyxin dependence. Deletion of astA, the first gene in the arginine degradation pathway, decreased phosphatidylglycerol and increased phosphatidylethanolamine levels in the outer membrane, thereby reducing the interaction with polymyxins. This study elucidates the molecular mechanism by which arginine metabolism impacts polymyxin dependence in A. baumannii, underscoring its critical role in improving diagnosis and treatment of life-threatening infections caused by "undetectable" polymyxin-dependent A. baumannii.

RIL-seq reveals extensive involvement of small RNAs in virulence and capsule regulation in hypervirulent Klebsiella pneumoniae.

Hypervirulent Klebsiella pneumoniae (hvKp) can infect healthy individuals, in contrast to classical strains that commonly cause nosocomial infections. The recent convergence of hypervirulence with carbapenem-resistance in K. pneumoniae can potentially create 'superbugs' that are challenging to treat. Understanding virulence regulation of hvKp is thus critical. Accumulating evidence suggest that posttranscriptional regulation by small RNAs (sRNAs) plays a role in bacterial virulence, but it has hardly been studied in K. pneumoniae. We applied RIL-seq to a prototypical clinical isolate of hvKp to unravel the Hfq-dependent RNA-RNA interaction (RRI) network. The RRI network is dominated by sRNAs, including predicted novel sRNAs, three of which we validated experimentally. We constructed a stringent subnetwork composed of RRIs that involve at least one hvKp virulence-associated gene and identified the capsule gene loci as a hub target where multiple sRNAs interact. We found that the sRNA OmrB suppressed both capsule production and hypermucoviscosity when overexpressed. Furthermore, OmrB base-pairs within kvrA coding region and partially suppresses translation of the capsule regulator KvrA. This agrees with current understanding of capsule as a major virulence and fitness factor. It emphasizes the intricate regulatory control of bacterial phenotypes by sRNAs, particularly of genes critical to bacterial physiology and virulence.

The TAM, a Translocation and Assembly Module for protein assembly and potential conduit for phospholipid transfer.

The assembly of ß-barrel proteins into the bacterial outer membrane is an essential process enabling the colonization of new environmental niches. The TAM was discovered as a module of the ß-barrel protein assembly machinery; it is a heterodimeric complex composed of an outer membrane protein (TamA) bound to an inner membrane protein (TamB). The TAM spans the periplasm, providing a scaffold through the peptidoglycan layer and catalyzing the translocation and assembly of ß-barrel proteins into the outer membrane. Recently, studies on another membrane protein (YhdP) have suggested that TamB might play a role in phospholipid transport to the outer membrane. Here we review and re-evaluate the literature covering the experimental studies on the TAM over the past decade, to reconcile what appear to be conflicting claims on the function of the TAM.

Dual recognition of multiple signals in bacterial outer membrane proteins enhances assembly and maintains membrane integrity.

Outer membrane proteins (OMPs) are essential components of the outer membrane of Gram-negative bacteria. In terms of protein targeting and assembly, the current dogma holds that a 'ß-signal' imprinted in the final ß-strand of the OMP engages the ß-barrel assembly machinery (BAM) complex to initiate membrane insertion and assembly of the OMP into the outer membrane. Here, we revealed an additional rule that signals equivalent to the ß-signal are repeated in other, internal ß-strands within bacterial OMPs, by peptidomimetic and mutational analysis. The internal signal is needed to promote the efficiency of the assembly reaction of these OMPs. BamD, an essential subunit of the BAM complex, recognizes the internal signal and the ß-signal, arranging several ß-strands and partial folding for rapid OMP assembly. The internal signal-BamD ordering system is not essential for bacterial viability but is necessary to retain the integrity of the outer membrane against antibiotics and other environmental insults.

The evolutionary mechanism of non-carbapenemase carbapenem-resistant phenotypes in Klebsiella spp.

Antibiotic resistance is driven by selection, but the degree to which a bacterial strain's evolutionary history shapes the mechanism and strength of resistance remains an open question. Here, we reconstruct the genetic and evolutionary mechanisms of carbapenem resistance in a clinical isolate of Klebsiella quasipneumoniae. A combination of short- and long-read sequencing, machine learning, and genetic and enzymatic analyses established that this carbapenem-resistant strain carries no carbapenemase-encoding genes. Genetic reconstruction of the resistance phenotype confirmed that two distinct genetic loci are necessary in order for the strain to acquire carbapenem resistance. Experimental evolution of the carbapenem-resistant strains in growth conditions without the antibiotic revealed that both loci confer a significant cost and are readily lost by de novo mutations resulting in the rapid evolution of a carbapenem-sensitive phenotype. To explain how carbapenem resistance evolves via multiple, low-fitness single-locus intermediates, we hypothesised that one of these loci had previously conferred adaptation to another antibiotic. Fitness assays in a range of drug concentrations show how selection in the antibiotic ceftazidime can select for one gene (blaDHA-1) potentiating the evolution of carbapenem resistance by a single mutation in a second gene (ompK36). These results show how a patient's treatment history might shape the evolution of antibiotic resistance and could explain the genetic basis of carbapenem-resistance found in many enteric-pathogens.

Epitopes in the capsular polysaccharide and the porin OmpK36 receptors are required for bacteriophage infection of Klebsiella pneumoniae.

To kill bacteria, bacteriophages (phages) must first bind to a receptor, triggering the release of the phage DNA into the bacterial cell. Many bacteria secrete polysaccharides that had been thought to shield bacterial cells from phage attack. We use a comprehensive genetic screen to distinguish that the capsule is not a shield but is instead a primary receptor enabling phage predation. Screening of a transposon library to select phage-resistant Klebsiella shows that the first receptor-binding event docks to saccharide epitopes in the capsule. We discover a second step of receptor binding, dictated by specific epitopes in an outer membrane protein. This additional and necessary event precedes phage DNA release to establish a productive infection. That such discrete epitopes dictate two essential binding events for phages has profound implications for understanding the evolution of phage resistance and what dictates host range, two issues critically important to translating knowledge of phage biology into phage therapies.

Surveying membrane landscapes: a new look at the bacterial cell surface.

Recent studies applying advanced imaging techniques are changing the way we understand bacterial cell surfaces, bringing new knowledge on everything from single-cell heterogeneity in bacterial populations to their drug sensitivity and mechanisms of antimicrobial resistance. In both Gram-positive and Gram-negative bacteria, the outermost surface of the bacterial cell is being imaged at nanoscale; as a result, topographical maps of bacterial cell surfaces can be constructed, revealing distinct zones and specific features that might uniquely identify each cell in a population. Functionally defined assembly precincts for protein insertion into the membrane have been mapped at nanoscale, and equivalent lipid-assembly precincts are suggested from discrete lipopolysaccharide patches. As we review here, particularly for Gram-negative bacteria, the applications of various modalities of nanoscale imaging are reawakening our curiosity about what is conceptually a 3D cell surface landscape: what it looks like, how it is made and how it provides resilience to respond to environmental impacts.

LPS O Antigen Plays a Key Role in Klebsiella pneumoniae Capsule Retention.

Despite the importance of encapsulation in bacterial pathogenesis, the biochemical mechanisms and forces that underpin retention of capsule by encapsulated bacteria are poorly understood. In Gram-negative bacteria, there may be interactions between lipopolysaccharide (LPS) core and capsule polymers, between capsule polymers with retained acyl carriers and the outer membrane, and in some bacteria, between the capsule polymers and Wzi, an outer membrane protein lectin. Our transposon studies in Klebsiella pneumoniae B5055 identified additional genes that, when insertionally inactivated, resulted in reduced encapsulation. Inactivation of the gene waaL, which encodes the ligase responsible for attaching the repeated O antigen of LPS to the LPS core, resulted in a significant reduction in capsule retention, measured by atomic force microscopy. This reduction in encapsulation was associated with increased sensitivity to human serum and decreased virulence in a murine model of respiratory infection and, paradoxically, with increased biofilm formation. The capsule in the WaaL mutant was physically smaller than that of the Wzi mutant of K. pneumoniae B5055. These results suggest that interactions between surface carbohydrate polymers may enhance encapsulation, a key phenotype in bacterial virulence, and provide another target for the development of antimicrobials that may avoid resistance issues associated with growth inhibition. IMPORTANCE Bacterial capsules, typically comprised of complex sugars, enable pathogens to avoid key host responses to infection, including phagocytosis. These capsules are synthesized within the bacteria, exported through the outer envelope, and then secured to the external surface of the organism by a force or forces that are incompletely described. This study shows that in the important hospital pathogen Klebsiella pneumoniae, the polysaccharide capsule is retained by interactions with other surface sugars, especially the repeated sugar molecule of the LPS molecule in Gram-negative bacteria known as "O antigen." This O antigen is joined to the LPS molecule by ligation, and loss of the enzyme responsible for ligation, a protein called WaaL, results in reduced encapsulation. Since capsules are essential to the virulence of many pathogens, WaaL might provide a target for new antimicrobial development, critical to the control of pathogens like K. pneumoniae that have become highly drug resistant.

A Structurally Characterized Staphylococcus aureus Evolutionary Escape Route from Treatment with the Antibiotic Linezolid.

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterial pathogen that presents great health concerns. Treatment requires the use of last-line antibiotics, such as members of the oxazolidinone family, of which linezolid is the first member to see regular use in the clinic. Here, we report a short time scale selection experiment in which strains of MRSA were subjected to linezolid treatment. Clonal isolates which had evolved a linezolid-resistant phenotype were characterized by whole-genome sequencing. Linezolid-resistant mutants were identified which had accumulated mutations in the ribosomal protein uL3. Multiple clones which had two mutations in uL3 exhibited resistance to linezolid, 2-fold higher than the clinical breakpoint. Ribosomes from this strain were isolated and subjected to single-particle cryo-electron microscopic analysis and compared to the ribosomes from the parent strain. We found that the mutations in uL3 lead to a rearrangement of a loop that makes contact with Helix 90, propagating a structural change over 15 Å away. This distal change swings nucleotide U2504 into the binding site of the antibiotic, causing linezolid resistance. IMPORTANCE Antibiotic resistance poses a critical problem to human health and decreases the utility of these lifesaving drugs. Of particular concern is the "superbug" methicillin-resistant Staphylococcus aureus (MRSA), for which treatment of infection requires the use of last-line antibiotics, including linezolid. In this paper, we characterize the atomic rearrangements which the ribosome, the target of linezolid, undergoes during its evolutionary journey toward becoming drug resistant. Using cryo-electron microscopy, we describe a particular molecular mechanism which MRSA uses to become resistant to linezolid.

Bacteriophages evolve enhanced persistence to a mucosal surface.

The majority of viruses within the gut are obligate bacterial viruses known as bacteriophages (phages). Their bacteriotropism underscores the study of phage ecology in the gut, where they modulate and coevolve with gut bacterial communities. Traditionally, these ecological and evolutionary questions were investigated empirically via in vitro experimental evolution and, more recently, in vivo models were adopted to account for physiologically relevant conditions of the gut. Here, we probed beyond conventional phage-bacteria coevolution to investigate potential tripartite evolutionary interactions between phages, their bacterial hosts, and the mammalian gut mucosa. To capture the role of the mammalian gut, we recapitulated a life-like gut mucosal layer using in vitro lab-on-a-chip devices (to wit, the gut-on-a-chip) and showed that the mucosal environment supports stable phage-bacteria coexistence. Next, we experimentally coevolved lytic phage populations within the gut-on-a-chip devices alongside their bacterial hosts. We found that while phages adapt to the mucosal environment via de novo mutations, genetic recombination was the key evolutionary force in driving mutational fitness. A single mutation in the phage capsid protein Hoc-known to facilitate phage adherence to mucus-caused altered phage binding to fucosylated mucin glycans. We demonstrated that the altered glycan-binding phenotype provided the evolved mutant phage a competitive fitness advantage over its ancestral wild-type phage in the gut-on-a-chip mucosal environment. Collectively, our findings revealed that phages-in addition to their evolutionary relationship with bacteria-are able to evolve in response to a mammalian-derived mucosal environment.

Tall tails: cryo-electron microscopy of phage tail DNA ejection conduits.

The majority of phages, viruses that infect prokaryotes, inject their genomic material into their host through a tubular assembly known as a tail. Despite the genomic diversity of tailed phages, only three morphological archetypes have been described: contractile tails of Myoviridae-like phages; short non-contractile tails of Podoviridae-like phages; and long and flexible non-contractile tails of Siphoviridae-like phages. While early cryo-electron microscopy (cryo-EM) work elucidated the organisation of the syringe-like injection mechanism of contractile tails, the intrinsic flexibility of the long non-contractile tails prevented high-resolution structural determination. In 2020, four cryo-EM structures of Siphoviridae-like tail tubes were solved and revealed common themes and divergences. The central tube is structurally conserved and homologous to the hexameric rings of the tail tube protein (TTP) also found in contractile tails, bacterial pyocins, and type VI secretion systems. The interior surface of the tube presents analogous motifs of negatively charged amino acids proposed to facilitate ratcheting of the DNA during genome ejection. The lack of a conformational change upon genome ejection implicates the tape measure protein in triggering genome release. A distinctive feature of Siphoviridae-like tails is their flexibility. This results from loose inter-ring connections that can asymmetrically stretch on one side to allow bending and flexing of the tube without breaking. The outer surface of the tube differs greatly and may be smooth or rugged due to additional Ig-like domains in TTP. Some of these variable domains may contribute to adsorption of the phage to prokaryotic and eukaryotic cell surfaces affecting tropism and virulence.

Adaptation of the periplasm to maintain spatial constraints essential for cell envelope processes and cell viability.

The cell envelope of Gram-negative bacteria consists of two membranes surrounding a periplasm and peptidoglycan layer. Molecular machines spanning the cell envelope depend on spatial constraints and load-bearing forces across the cell envelope and surface. The mechanisms dictating spatial constraints across the cell envelope remain incompletely defined. In Escherichia coli, the coiled-coil lipoprotein Lpp contributes the only covalent linkage between the outer membrane and the underlying peptidoglycan layer. Using proteomics, molecular dynamics, and a synthetic lethal screen, we show that lengthening Lpp to the upper limit does not change the spatial constraint but is accommodated by other factors which thereby become essential for viability. Our findings demonstrate E. coli expressing elongated Lpp does not simply enlarge the periplasm in response, but the bacteria accommodate by a combination of tilting Lpp and reducing the amount of the covalent bridge. By genetic screening, we identified all of the genes in E. coli that become essential in order to enact this adaptation, and by quantitative proteomics discovered that very few proteins need to be up- or down-regulated in steady-state levels in order to accommodate the longer Lpp. We observed increased levels of factors determining cell stiffness, a decrease in membrane integrity, an increased membrane vesiculation and a dependance on otherwise non-essential tethers to maintain lipid transport and peptidoglycan biosynthesis. Further this has implications for understanding how spatial constraint across the envelope controls processes such as flagellum-driven motility, cellular signaling, and protein translocation.

A noncanonical chaperone interacts with drug efflux pumps during their assembly into bacterial outer membranes.

Bacteria have membrane-spanning efflux pumps to secrete toxic compounds ranging from heavy metal ions to organic chemicals, including antibiotic drugs. The overall architecture of these efflux pumps is highly conserved: with an inner membrane energy-transducing subunit coupled via an adaptor protein to an outer membrane conduit subunit that enables toxic compounds to be expelled into the environment. Here, we map the distribution of efflux pumps across bacterial lineages to show these proteins are more widespread than previously recognised. Complex phylogenetics support the concept that gene cassettes encoding the subunits for these pumps are commonly acquired by horizontal gene transfer. Using TolC as a model protein, we demonstrate that assembly of conduit subunits into the outer membrane uses the chaperone TAM to physically organise the membrane-embedded staves of the conduit subunit of the efflux pump. The characteristics of this assembly pathway have impact for the acquisition of efflux pumps across bacterial species and for the development of new antimicrobial compounds that inhibit efflux pump function.

Targeting bacterial outer-membrane remodelling to impact antimicrobial drug resistance.

The cell envelope is essential for survival and adaptation of bacteria. Bacterial membrane proteins include the major porins that mediate the influx of nutrients and several classes of antimicrobial drugs. Consequently, membrane remodelling is closely linked to antimicrobial resistance (AMR). Knowledge of bacterial membrane protein biogenesis and turnover underpins our understanding of bacterial membrane remodelling and the consequences that this process have in the evolution of AMR phenotypes. At the population level, the evolution of phenotypes is a reversible process, and we can use these insights to deploy evolutionary principles to resensitize bacteria to existing antimicrobial drugs. In our opinion, fundamental knowledge is opening a new way of thinking towards sustainable solutions to the mounting crisis in AMR. Here we discuss what is known about outer-membrane remodelling in bacteria and how the process could be targeted as a means to restore sensitivity to antimicrobial drugs. Bacteriophages are highlighted as a powerful means to exert this control over membrane remodelling but they require careful selection so as to reverse, and not exacerbate, AMR phenotypes.

Neisseria gonorrhoeae-derived outer membrane vesicles package ß-lactamases to promote antibiotic resistance.

Neisseria gonorrhoeae causes the sexually transmitted disease gonorrhoea. The treatment of gonorrhoea is becoming increasingly challenging, as N. gonorrhoeae has developed resistance to antimicrobial agents routinely used in the clinic. Resistance to penicillin is wide-spread partly due to the acquisition of ß-lactamase genes. How N. gonorrhoeae survives an initial exposure to ß-lactams before acquiring resistance genes remains to be understood. Here, using a panel of clinical isolates of N. gonorrhoeae we show that the ß-lactamase enzyme is packaged into outer membrane vesicles (OMVs) by strains expressing blaTEM-1B or blaTEM-106, which protects otherwise susceptible clinical isolates from the ß-lactam drug amoxycillin. We characterized the phenotypes of these clinical isolates of N. gonorrhoeae and the time courses over which the cross-protection of the strains is effective. Imaging and biochemical assays suggest that OMVs promote the transfer of proteins and lipids between bacteria. Thus, N. gonorrhoeae strains secret antibiotic degrading enzymes via OMVs enabling survival of otherwise susceptible bacteria.

Mechanistic Insights into the Capsule-Targeting Depolymerase from a Klebsiella pneumoniae Bacteriophage.

The production of capsular polysaccharides by Klebsiella pneumoniae protects the bacterial cell from harmful environmental factors such as antimicrobial compounds and infection by bacteriophages (phages). To bypass this protective barrier, some phages encode polysaccharide-degrading enzymes referred to as depolymerases to provide access to cell surface receptors. Here, we characterized the phage RAD2, which infects K. pneumoniae strains that produce the widespread, hypervirulence-associated K2-type capsular polysaccharide. Using transposon-directed insertion sequencing, we have shown that the production of capsule is an absolute requirement for efficient RAD2 infection by serving as a first-stage receptor. We have identified the depolymerase responsible for recognition and degradation of the capsule, determined that the depolymerase forms globular appendages on the phage virion tail tip, and present the cryo-electron microscopy structure of the RAD2 capsule depolymerase at 2.7-Å resolution. A putative active site for the enzyme was identified, comprising clustered negatively charged residues that could facilitate the hydrolysis of target polysaccharides. Enzymatic assays coupled with mass spectrometric analyses of digested oligosaccharide products provided further mechanistic insight into the hydrolase activity of the enzyme, which, when incubated with K. pneumoniae, removes the capsule and sensitizes the cells to serum-induced killing. Overall, these findings expand our understanding of how phages target the Klebsiella capsule for infection, providing a framework for the use of depolymerases as antivirulence agents against this medically important pathogen. IMPORTANCE Klebsiella pneumoniae is a medically important pathogen that produces a thick protective capsule that is essential for pathogenicity. Phages are natural predators of bacteria, and many encode diverse "capsule depolymerases" which specifically degrade the capsule of their hosts, an exploitable trait for potential therapies. We have determined the first structure of a depolymerase that targets the clinically relevant K2 capsule and have identified its putative active site, providing hints to its mechanism of action. We also show that Klebsiella cells treated with a recombinant form of the depolymerase are stripped of capsule, inhibiting their ability to grow in the presence of serum, demonstrating the anti-infective potential of these robust and readily producible enzymes against encapsulated bacterial pathogens such as K. pneumoniae.