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1.
PLoS Pathog ; 19(5): e1011367, 2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37146068

RESUMO

Klebsiella pneumoniae presents as two circulating pathotypes: classical K. pneumoniae (cKp) and hypervirulent K. pneumoniae (hvKp). Classical isolates are considered urgent threats due to their antibiotic resistance profiles, while hvKp isolates have historically been antibiotic susceptible. Recently, however, increased rates of antibiotic resistance have been observed in both hvKp and cKp, further underscoring the need for preventive and effective immunotherapies. Two distinct surface polysaccharides have gained traction as vaccine candidates against K. pneumoniae: capsular polysaccharide and the O-antigen of lipopolysaccharide. While both targets have practical advantages and disadvantages, it remains unclear which of these antigens included in a vaccine would provide superior protection against matched K. pneumoniae strains. Here, we report the production of two bioconjugate vaccines, one targeting the K2 capsular serotype and the other targeting the O1 O-antigen. Using murine models, we investigated whether these vaccines induced specific antibody responses that recognize K2:O1 K. pneumoniae strains. While each vaccine was immunogenic in mice, both cKp and hvKp strains exhibited decreased O-antibody binding in the presence of capsule. Further, O1 antibodies demonstrated decreased killing in serum bactericidal assays with encapsulated strains, suggesting that the presence of K. pneumoniae capsule blocks O1-antibody binding and function. Finally, the K2 vaccine outperformed the O1 vaccine against both cKp and hvKp in two different murine infection models. These data suggest that capsule-based vaccines may be superior to O-antigen vaccines for targeting hvKp and some cKp strains, due to capsule blocking the O-antigen.


Assuntos
Infecções por Klebsiella , Vacinas , Camundongos , Animais , Virulência , Antígenos O , Klebsiella pneumoniae , Lipopolissacarídeos/metabolismo , Antibacterianos/farmacologia , Infecções por Klebsiella/prevenção & controle
2.
J Infect Dis ; 230(3): 578-589, 2024 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-38401891

RESUMO

Klebsiella pneumoniae is the leading cause of neonatal sepsis and is increasingly difficult to treat owing to antibiotic resistance. Vaccination represents a tractable approach to combat this resistant bacterium; however, there is currently not a licensed vaccine. Surface polysaccharides, including O-antigens of lipopolysaccharide, have long been attractive candidates for vaccine inclusion. Herein we describe the generation of a bioconjugate vaccine targeting 7 predominant O-antigen subtypes in K. pneumoniae. Each bioconjugate was immunogenic in isolation, with limited cross-reactivity among subtypes. Vaccine-induced antibodies demonstrated varying degrees of binding to a wide variety of K. pneumoniae strains. Furthermore, serum from vaccinated mice induced complement-mediated killing of many of these strains. Finally, increased capsule interfered with the ability of O-antigen antibodies to bind and mediate killing of some K. pneumoniae strains. Taken together, these data indicate that this novel heptavalent O-antigen bioconjugate vaccine formulation exhibits limited efficacy against some, but not all, K. pneumoniae isolates.


Assuntos
Anticorpos Antibacterianos , Vacinas Bacterianas , Infecções por Klebsiella , Klebsiella pneumoniae , Antígenos O , Klebsiella pneumoniae/imunologia , Antígenos O/imunologia , Antígenos O/química , Animais , Anticorpos Antibacterianos/imunologia , Infecções por Klebsiella/imunologia , Infecções por Klebsiella/prevenção & controle , Infecções por Klebsiella/microbiologia , Vacinas Bacterianas/imunologia , Camundongos , Feminino , Vacinas Conjugadas/imunologia , Camundongos Endogâmicos BALB C , Humanos
3.
Glycobiology ; 33(1): 57-74, 2023 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-36239418

RESUMO

Bacterial protein glycosylation is commonly mediated by oligosaccharyltransferases (OTases) that transfer oligosaccharides en bloc from preassembled lipid-linked precursors to acceptor proteins. Natively, O-linking OTases usually transfer a single repeat unit of the O-antigen or capsular polysaccharide to the side chains of serine or threonine on acceptor proteins. Three major families of bacterial O-linking OTases have been described: PglL, PglS, and TfpO. TfpO is limited to transferring short oligosaccharides both in its native context and when heterologously expressed in glycoengineered Escherichia coli. On the other hand, PglL and PglS can transfer long-chain polysaccharides when expressed in glycoengineered E. coli. Herein, we describe the discovery and functional characterization of a novel family of bacterial O-linking OTases termed TfpM from Moraxellaceae bacteria. TfpM proteins are similar in size and sequence to TfpO enzymes but can transfer long-chain polysaccharides to acceptor proteins. Phylogenetic analyses demonstrate that TfpM proteins cluster in distinct clades from known bacterial OTases. Using a representative TfpM enzyme from Moraxella osloensis, we determined that TfpM glycosylates a C-terminal threonine of its cognate pilin-like protein and identified the minimal sequon required for glycosylation. We further demonstrated that TfpM has broad substrate tolerance and can transfer diverse glycans including those with glucose, galactose, or 2-N-acetyl sugars at the reducing end. Last, we find that a TfpM-derived bioconjugate is immunogenic and elicits serotype-specific polysaccharide IgG responses in mice. The glycan substrate promiscuity of TfpM and identification of the minimal TfpM sequon renders this enzyme a valuable additional tool for expanding the glycoengineering toolbox.


Assuntos
Hexosiltransferases , Moraxellaceae , Animais , Camundongos , Moraxellaceae/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Filogenia , Hexosiltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Fímbrias , Polissacarídeos/metabolismo , Bactérias/metabolismo
4.
Proc Natl Acad Sci U S A ; 116(37): 18655-18663, 2019 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-31455739

RESUMO

Hypervirulent Klebsiella pneumoniae (hvKp) is globally disseminating as a community-acquired pathogen causing life-threatening infections in healthy individuals. The fact that a dose as little as 50 bacteria is lethal to mice illustrates the dramatic increase of virulence associated with hvKp strains compared with classical K. pneumoniae (cKp) strains, which require lethal doses greater than 107 bacteria. Until recently, these virulent strains were mostly antibiotic-susceptible. However, multidrug-resistant (MDR) hvKp strains have been emerging, spawning a new generation of hypervirulent "superbugs." The mechanisms of hypervirulence are not fully defined, but overproduction of capsular polysaccharide significantly impedes host clearance, resulting in increased pathogenicity of hvKp strains. While there are more than 80 serotypes of K. pneumoniae, the K1 and K2 serotypes cause the vast majority of hypervirulent infections. Therefore, a glycoconjugate vaccine targeting these 2 serotypes could significantly reduce hvKp infection. Conventionally, glycoconjugate vaccines are manufactured using intricate chemical methodologies to covalently attach purified polysaccharides to carrier proteins, which is widely considered to be technically challenging. Here we report on the recombinant production and analytical characterization of bioconjugate vaccines, enzymatically produced in glycoengineered Escherichia coli cells, against the 2 predominant hypervirulent K. pneumoniae serotypes, K1 and K2. The K. pneumoniae bioconjugates are immunogenic and efficacious, protecting mice against lethal infection from 2 hvKp strains, NTUH K-2044 and ATCC 43816. This preclinical study constitutes a key step toward preventing further global dissemination of hypervirulent MDR hvKp strains.


Assuntos
Vacinas Bacterianas/imunologia , Infecções Comunitárias Adquiridas/prevenção & controle , Infecções por Klebsiella/prevenção & controle , Klebsiella pneumoniae/imunologia , Fatores de Virulência/imunologia , Animais , Vacinas Bacterianas/administração & dosagem , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Infecções Comunitárias Adquiridas/imunologia , Infecções Comunitárias Adquiridas/microbiologia , Modelos Animais de Doenças , Farmacorresistência Bacteriana Múltipla , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Humanos , Imunogenicidade da Vacina , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/patogenicidade , Lipopolissacarídeos/genética , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/metabolismo , Engenharia Metabólica , Camundongos , Vacinas Conjugadas/administração & dosagem , Vacinas Conjugadas/imunologia , Virulência/imunologia , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
5.
Glycobiology ; 31(9): 1192-1203, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-33997889

RESUMO

Bioconjugate vaccines, consisting of polysaccharides attached to carrier proteins, are enzymatically generated using prokaryotic glycosylation systems in a process termed bioconjugation. Key to bioconjugation are a group of enzymes known as oligosaccharyltransferases (OTases) that transfer polysaccharides to engineered carrier proteins containing conserved amino acid sequences known as sequons. The most recently discovered OTase, PglS, has been shown to have the broadest substrate scope, transferring many different types of bacterial glycans including those with glucose at the reducing end. However, PglS is currently the least understood in terms of the sequon it recognizes. PglS is a pilin-specific O-linking OTase that naturally glycosylates a single protein, ComP. In addition to ComP, we previously demonstrated that an engineered carrier protein containing a large fragment of ComP is also glycosylated by PglS. Here we sought to identify the minimal ComP sequon sufficient for PglS glycosylation. We tested >100 different ComP fragments individually fused to Pseudomonas aeruginosa exotoxin A (EPA), leading to the identification of an 11-amino acid sequence sufficient for robust glycosylation by PglS. We also demonstrate that the placement of the ComP sequon on the carrier protein is critical for stability and subsequent glycosylation. Moreover, we identify novel sites on the surface of EPA that are amenable to ComP sequon insertion and find that Cross-Reactive Material 197 fused to a ComP fragment is also glycosylated. These results represent a significant expansion of the glycoengineering toolbox as well as our understanding of bacterial O-linking sequons.


Assuntos
Hexosiltransferases , Sequência de Aminoácidos , Proteínas de Fímbrias/metabolismo , Glicosilação , Hexosiltransferases/metabolismo , Proteínas de Membrana
6.
Glycobiology ; 29(7): 519-529, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-30989179

RESUMO

The first, general glycosylation pathway in bacteria, the N-linked glycosylation system of Campylobacter jejuni, was discovered two decades ago. Since then, many diverse prokaryotic glycosylation systems have been characterized, including O-linked glycosylation systems that have no homologous counterparts in eukaryotic organisms. Shortly after these discoveries, glycosylation pathways were recombinantly introduced into E. coli creating the field of bacterial glycoengineering. Bacterial glycoengineering is an emerging biotechnological tool that harnesses prokaryotic glycosylation systems for the generation of recombinantly glycosylated proteins using E. coli as a host. Over the last decade, as our understanding of prokaryotic glycosylation systems has advanced, so too has the glycoengineering toolbox. Currently, glycoengineering utilizes two broad approaches to recombinantly glycosylate proteins, both of which can generate N- or O-linkages: oligosaccharyltransferase (OTase)-dependent and OTase-independent. This review discusses the applications of these bacterial glycoengineering techniques as they relate to the development of glycoconjugate vaccines, therapeutic proteins, and diagnostics.


Assuntos
Escherichia coli/genética , Escherichia coli/metabolismo , Glicoconjugados/metabolismo , Engenharia Metabólica , Vacinas/metabolismo , Campylobacter jejuni/metabolismo , Configuração de Carboidratos , Glicoconjugados/genética , Glicosilação , Vacinas/genética
7.
J Biol Chem ; 292(22): 9075-9087, 2017 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-28373284

RESUMO

Pathogenic Acinetobacter species, including Acinetobacter baumannii and Acinetobacter nosocomialis, are opportunistic human pathogens of increasing relevance worldwide. Although their mechanisms of drug resistance are well studied, the virulence factors that govern Acinetobacter pathogenesis are incompletely characterized. Here we define the complete secretome of A. nosocomialis strain M2 in minimal medium and demonstrate that pathogenic Acinetobacter species produce both a functional type I secretion system (T1SS) and a contact-dependent inhibition (CDI) system. Using bioinformatics, quantitative proteomics, and mutational analyses, we show that Acinetobacter uses its T1SS for exporting two putative T1SS effectors, an Repeats-in-Toxin (RTX)-serralysin-like toxin, and the biofilm-associated protein (Bap). Moreover, we found that mutation of any component of the T1SS system abrogated type VI secretion activity under nutrient-limited conditions, indicating a previously unrecognized cross-talk between these two systems. We also demonstrate that the Acinetobacter T1SS is required for biofilm formation. Last, we show that both A. nosocomialis and A. baumannii produce functioning CDI systems that mediate growth inhibition of sister cells lacking the cognate immunity protein. The Acinetobacter CDI systems are widely distributed across pathogenic Acinetobacter species, with many A. baumannii isolates harboring two distinct CDI systems. Collectively, these data demonstrate the power of differential, quantitative proteomics approaches to study secreted proteins, define the role of previously uncharacterized protein export systems, and observe cross-talk between secretion systems in the pathobiology of medically relevant Acinetobacter species.


Assuntos
Acinetobacter baumannii/metabolismo , Acinetobacter baumannii/patogenicidade , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/metabolismo , Acinetobacter baumannii/genética , Proteínas de Bactérias/genética , Sistemas de Secreção Bacterianos/genética , Humanos
8.
Glycobiology ; 28(7): 522-533, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29668902

RESUMO

Acinetobacter baumannii is an opportunistic human pathogen with the highest reported rates of multidrug resistance among Gram-negative pathogens. The capsular polysaccharide of A. baumannii is considered one of its most significant virulence factors providing resistance against complemented-mediated killing. Capsule synthesis in A. baumannii is usually initiated by the phosphoglycosyltransferase PglC. PglC transfers a phosphosugar from a nucleotide diphosphate-sugar to a polyprenol phosphate generating a polyprenol diphosphate-linked monosaccharide. Traditionally, PglC was thought to have stringent specificity towards UDP-N-N'-diacetylbacillosamine (UDP-diNAcBac). In this work we demonstrate that A. baumannii PglC has the ability to utilize three different UDP-sugar substrates: UDP-N-acetylglucosamine (UDP-GlcNAc), UDP-N-acetylgalactosamine (UDP-GalNAc) or UDP-diNAcBac. Using phylogenetic analyses, we first demonstrate that A. baumannii PglC orthologs separate into three distinct clades. Moreover, all members within a clade are predicted to have the same preference for one of the three possible sugar substrates. To experimentally determine the substrate specificity of each clade, we utilized in vivo complementation models and NMR analysis. We demonstrate that UDP-diNAcBac is accommodated by all PglC orthologs, but some orthologs evolved to utilize UDP-GlcNAc or UDP-GalNAc in a clade-dependent manner. Furthermore, we show that a single point mutation can modify the sugar specificity of a PglC ortholog specific for UDP-diNAcBac and that introduction of a non-native PglC ortholog into A. baumannii can generate a new capsule serotype. Collectively, these studies begin to explain why A. baumannii strains have such highly diverse glycan repertoires.


Assuntos
Acinetobacter baumannii/enzimologia , Proteínas de Bactérias/química , Glucosiltransferases/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Mutação , Ligação Proteica , Especificidade por Substrato , Uridina Difosfato N-Acetilglicosamina/metabolismo
9.
PLoS Pathog ; 12(1): e1005391, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26764912

RESUMO

Acinetobacter baumannii, A. nosocomialis, and A. pittii have recently emerged as opportunistic human pathogens capable of causing severe human disease; however, the molecular mechanisms employed by Acinetobacter to cause disease remain poorly understood. Many pathogenic members of the genus Acinetobacter contain genes predicted to encode proteins required for the biogenesis of a type II secretion system (T2SS), which have been shown to mediate virulence in many Gram-negative organisms. Here we demonstrate that Acinetobacter nosocomialis strain M2 produces a functional T2SS, which is required for full virulence in both the Galleria mellonella and murine pulmonary infection models. Importantly, this is the first bona fide secretion system shown to be required for virulence in Acinetobacter. Using bioinformatics, proteomics, and mutational analyses, we show that Acinetobacter employs its T2SS to export multiple substrates, including the lipases LipA and LipH as well as the protease CpaA. Furthermore, the Acinetobacter T2SS, which is found scattered amongst five distinct loci, does not contain a dedicated pseudopilin peptidase, but instead relies on the type IV prepilin peptidase, reinforcing the common ancestry of these two systems. Lastly, two of the three secreted proteins characterized in this study require specific chaperones for secretion. These chaperones contain an N-terminal transmembrane domain, are encoded adjacently to their cognate effector, and their disruption abolishes type II secretion of their cognate effector. Bioinformatic analysis identified putative chaperones located adjacent to multiple previously known type II effectors from several Gram-negative bacteria, which suggests that T2SS chaperones constitute a separate class of membrane-associated chaperones mediating type II secretion.


Assuntos
Infecções por Acinetobacter/metabolismo , Acinetobacter/patogenicidade , Chaperonas Moleculares/metabolismo , Sistemas de Secreção Tipo II/metabolismo , Animais , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Virulência
10.
J Biol Chem ; 291(44): 22924-22935, 2016 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-27634041

RESUMO

Acinetobacter baumannii is a Gram-negative coccobacillus found primarily in hospital settings that has recently emerged as a source of hospital-acquired infections. A. baumannii expresses a variety of virulence factors, including type IV pili, bacterial extracellular appendages often essential for attachment to host cells. Here, we report the high resolution structures of the major pilin subunit, PilA, from three Acinetobacter strains, demonstrating that A. baumannii subsets produce morphologically distinct type IV pilin glycoproteins. We examine the consequences of this heterogeneity for protein folding and assembly as well as host-cell adhesion by Acinetobacter Comparisons of genomic and structural data with pilin proteins from other species of soil gammaproteobacteria suggest that these structural differences stem from evolutionary pressure that has resulted in three distinct classes of type IVa pilins, each found in multiple species.


Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Proteínas de Bactérias/química , Fímbrias Bacterianas/química , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/química , Acinetobacter baumannii/classificação , Acinetobacter baumannii/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana Múltipla , Evolução Molecular , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/metabolismo , Gammaproteobacteria/química , Gammaproteobacteria/classificação , Gammaproteobacteria/isolamento & purificação , Gammaproteobacteria/metabolismo , Regulação Bacteriana da Expressão Gênica , Humanos , Modelos Moleculares , Filogenia , Microbiologia do Solo
11.
Mol Microbiol ; 96(5): 1023-41, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25727908

RESUMO

Multiple species within the Acinetobacter genus are nosocomial opportunistic pathogens of increasing relevance worldwide. Among the virulence factors utilized by these bacteria are the type IV pili and a protein O-glycosylation system. Glycosylation is mediated by O-oligosaccharyltransferases (O-OTases), enzymes that transfer the glycan from a lipid carrier to target proteins. O-oligosaccharyltransferases are difficult to identify due to similarities with the WaaL ligases that catalyze the last step in lipopolysaccharide synthesis. A bioinformatics analysis revealed the presence of two genes encoding putative O-OTases or WaaL ligases in most of the strains within the genus Acinetobacter. Employing A. nosocomialis M2 and A. baylyi ADP1 as model systems, we show that these genes encode two O-OTases, one devoted uniquely to type IV pilin, and the other one responsible for glycosylation of multiple proteins. With the exception of ADP1, the pilin-specific OTases in Acinetobacter resemble the TfpO/PilO O-OTase from Pseudomonas aeruginosa. In ADP1 instead, the two O-OTases are closely related to PglL, the general O-OTase first discovered in Neisseria. However, one of them is exclusively dedicated to the glycosylation of the pilin-like protein ComP. Our data reveal an intricate and remarkable evolutionary pathway for bacterial O-OTases and provide novel tools for glycoengineering.


Assuntos
Acinetobacter/enzimologia , Acinetobacter/genética , Proteínas de Bactérias/metabolismo , Proteínas de Fímbrias/metabolismo , Glicosiltransferases/metabolismo , Hexosiltransferases/metabolismo , Ligases/metabolismo , Proteínas de Membrana/metabolismo , Acinetobacter/patogenicidade , Proteínas de Bactérias/genética , Biologia Computacional , Proteínas de Fímbrias/genética , Glicopeptídeos/química , Glicosilação , Glicosiltransferases/genética , Hexosiltransferases/genética , Proteínas de Membrana/genética , Neisseria/metabolismo , Polissacarídeos/metabolismo
12.
BMC Infect Dis ; 16: 346, 2016 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-27449800

RESUMO

BACKGROUND: Acinetobacter species are associated with increasing mortality due to emerging drug-resistance. Pediatric Acinetobacter infections are largely undefined in developed countries and clinical laboratory identification methods do not reliably differentiate between members of the Acinetobacter calcoaceticus-baumannii complex, leading to improper identification. Therefore we aimed to determine risk factors for invasive Acinetobacter infections within an academic, pediatric setting as well as defining microbiologic characteristics of predominant strains. METHODS: Twenty-four invasive Acinetobacter isolates were collected from 2009-2013. Comparative sequence analysis of the rpoB gene was performed coupled with phenotypic characterization of antibiotic resistance, motility, biofilm production and clinical correlation. RESULTS: Affected patients had a median age of 3.5 years, and 71 % had a central catheter infection source. rpoB gene sequencing revealed a predominance of A. pittii (45.8 %) and A. baumannii (33.3 %) strains. There was increasing incidence of A. pittii over the study. Two fatalities occurred in the A. pittii group. Seventeen percent of isolates were multi-drug resistant. A pittii and A. baumannii strains were similar in motility, but A pittii strains had significantly more biofilm production (P value = 0.018). CONCLUSIONS: A. pittii was the most isolated species highlighting the need for proper species identification. The isolated strains had limited acute mortality in children, but the occurrence of more multi-drug resistant strains in the future is a distinct possibility, justifying continued research and accurate species identification.


Assuntos
Infecções por Acinetobacter/microbiologia , Acinetobacter/isolamento & purificação , Infecção Hospitalar/microbiologia , Centros Médicos Acadêmicos , Acinetobacter/efeitos dos fármacos , Acinetobacter/genética , Acinetobacter/fisiologia , Infecções por Acinetobacter/diagnóstico , Infecções por Acinetobacter/tratamento farmacológico , Infecções por Acinetobacter/etiologia , Adolescente , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Biofilmes , Criança , Pré-Escolar , Infecção Hospitalar/diagnóstico , Infecção Hospitalar/tratamento farmacológico , Infecção Hospitalar/etiologia , Farmacorresistência Bacteriana Múltipla , Feminino , Hospitais Pediátricos , Humanos , Lactente , Recém-Nascido , Masculino , Estudos Retrospectivos , Fatores de Risco , Adulto Jovem
13.
J Bacteriol ; 198(6): 880-7, 2015 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-26712938

RESUMO

The genus Acinetobacter encompasses multiple nosocomial opportunistic pathogens that are of increasing worldwide relevance because of their ability to survive exposure to various antimicrobial and sterilization agents. Among these, Acinetobacter baumannii, Acinetobacter nosocomialis, and Acinetobacter pittii are the most frequently isolated in hospitals around the world. Despite the growing incidence of multidrug-resistant Acinetobacter spp., little is known about the factors that contribute to pathogenesis. New strategies for treating and managing infections caused by multidrug-resistant Acinetobacter strains are urgently needed, and this requires a detailed understanding of the pathobiology of these organisms. In recent years, some virulence factors important for Acinetobacter colonization have started to emerge. In this review, we focus on several recently described virulence factors that act at the bacterial surface level, such as the capsule, O-linked protein glycosylation, and adhesins. Furthermore, we describe the current knowledge regarding the type II and type VI secretion systems present in these strains.


Assuntos
Infecções por Acinetobacter/microbiologia , Acinetobacter/patogenicidade , Infecção Hospitalar/microbiologia , Farmacorresistência Bacteriana Múltipla , Fatores de Virulência/metabolismo , Acinetobacter/efeitos dos fármacos , Infecções por Acinetobacter/epidemiologia , Adesinas Bacterianas/metabolismo , Cápsulas Bacterianas/metabolismo , Sistemas de Secreção Bacterianos , Infecção Hospitalar/epidemiologia , Humanos , Antígenos O/metabolismo
14.
bioRxiv ; 2024 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-38746292

RESUMO

Background: Klebsiella pneumonia (Kpn) is the fourth leading cause of infection-related deaths globally, yet little is known about human antibody responses to invasive Kpn. In this study, we sought to determine whether the O-specific polysaccharide (OPS) antigen, a vaccine candidate, is immunogenic in humans with Kpn bloodstream infection (BSI). We also sought to define the cross-reactivity of human antibody responses among structurally related Kpn OPS subtypes and to assess the impact of capsule production on OPS-targeted antibody binding and function. Methods: We measured plasma antibody responses to OPS (and MrkA, a fimbrial protein) in a cohort of patients with Kpn BSI and compared these with controls, including a cohort of healthy individuals and a cohort of individuals with Enterococcus BSI. We performed flow cytometry to measure the impact of Kpn capsule production on whole cell antibody binding and complement deposition, utilizing patient isolates with variable levels of capsule production and isogenic capsule-deficient strains derived from these isolates. Findings: We enrolled 69 patients with Kpn BSI. Common OPS serotypes accounted for 57/69 (83%) of infections. OPS was highly immunogenic in patients with Kpn BSI, and peak OPS-IgG antibody responses in patients were 10 to 30-fold higher than antibody levels detected in healthy controls, depending on the serotype. There was significant cross-reactivity among structurally similar OPS subtypes, including the O1v1/O1v2, O2v1/O2v2 and O3/O3b subtypes. Physiological amounts of capsule produced by both hyperencapsulated and non-hyperencapsulated Kpn significantly inhibited OPS-targeted antibody binding and function. Interpretation: OPS was highly immunogenic in patients with Kpn BSI, supporting its potential as a candidate vaccine antigen. The strong cross-reactivity observed between similar OPS subtypes in humans with Kpn BSI suggests that it may not be necessary to include all subtypes in an OPS-based vaccine. However, these observations are tempered by the fact that capsule production, even in non-highly encapsulated strains, has the potential to interfere with OPS antibody binding. This may limit the effectiveness of vaccines that exclusively target OPS. Funding: National Institute of Allergy and Infectious Diseases at the National Institutes of Health. Research in Context: Evidence before this study: Despite the potential of O-specific polysaccharide (OPS) as a vaccine antigen against Klebsiella pneumoniae (Kpn), the immunogenicity of OPS in humans remains largely unstudied, creating a significant knowledge gap with regard to vaccine development. A search of PubMed for publications up to March 18, 2024, using the terms " Klebsiella pneumoniae " and "O-specific polysaccharide" or "O-antigen" or "lipopolysaccharide" revealed no prior studies addressing OPS antibody responses in humans with Kpn bloodstream infections (BSI). One prior study 1 evaluated antibody response to a single lipopolysaccharide (which contains one subtype of OPS) in humans with invasive Kpn infection; however, in this study OPS typing of the infecting strains and target antigen were not described. Added value of this study: Our investigation into OPS immunogenicity in a human cohort marks a significant advance. Analyzing plasma antibody responses in 69 patients with Kpn BSI, we found OPS to be broadly immunogenic across all the types and subtypes examined, and there was significant cross-reactivity among structurally related OPS antigens. We also demonstrated that Kpn capsule production inhibit OPS antibody binding and the activation of complement on the bacterial surface, even in classical Kpn strains expressing lower levels of capsule.Implications of all the available evidence: While the immunogenicity and broad cross-reactivity of OPS in humans with Kpn BSI suggests it is a promising vaccine candidate, the obstruction of OPS antibody binding and engagement by physiologic levels of Kpn capsule underscores the potential limitations of an exclusively OPS-antigen based vaccine for Kpn. Our study provides insights for the strategic development of vaccines aimed at combating Kpn infections, an important antimicrobial resistant pathogen.

15.
bioRxiv ; 2023 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-38168360

RESUMO

Klebsiella pneumoniae is a concerning pathogen that is now the leading cause of neonatal sepsis and is increasingly difficult to treat due to heightened antibiotic resistance. Thus, there is an urgent need for preventive and effective immunotherapies targeting K. pneumoniae. Vaccination represents a tractable approach to combat this resistant bacterium in some settings; however, there is currently not a licensed K. pneumoniae vaccine available. K. pneumoniae surface polysaccharides, including the terminal O-antigen polysaccharides of lipopolysaccharide, have long been attractive candidates for vaccine inclusion. Herein we describe the generation of a bioconjugate vaccine targeting seven of the predominant O-antigen subtypes in K. pneumoniae. Each of the seven bioconjugates were immunogenic in isolation, with limited cross-reactivity among subtypes. Vaccine-induced antibodies demonstrated varying degrees of binding to a wide variety of K. pneumoniae strains, including suspected hypervirulent strains, all expressing different O-antigen and capsular polysaccharide combinations. Further, sera from vaccinated mice induced complement-mediated killing of many of these K. pneumoniae strains. Finally, we found that increased quantity of capsule interferes with O-antigen antibodies' ability to bind and mediate killing of some K. pneumoniae strains, including those carrying hypervirulence-associated genes. Taken together, these data indicate that this novel heptavalent O-antigen bioconjugate vaccine formulation exhibits promising efficacy against some, but not all, K. pneumoniae isolates.

16.
Vaccine ; 40(42): 6107-6113, 2022 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-36115800

RESUMO

Capsular polysaccharides (CPSs), with which most pathogenic bacterial surfaces are decorated, have been used as the main components of glycoconjugate vaccines against bacterial diseases in clinical practice worldwide. Pneumococcal conjugate vaccines (PCVs) are administered globally to prevent invasive pneumococcal disease (IPD). While PCVs have played important roles in controlling IPD in all age groups, their empirical, and labor-intensive chemical conjugation yield poorly characterized, heterogeneous, and variably immunogenic vaccines, with poor immune responses in high-risk populations such as the elderly and patients with weak immune systems. We previously developed a method that bypasses the dependency of chemical conjugation and instead exploits prokaryotic glycosylation systems to produce pneumococcal conjugate vaccines. The bioconjugation platform relies on a conjugating enzyme to transfer a bacterial polysaccharide to an engineered carrier protein all within the lab safe bacterium E. coli. In these studies, we demonstrate that a serotype 8 pneumococcal bioconjugate vaccine is highly immunogenic and elicits functionally protective anti-serotype 8 antibody responses. Specifically, using multiple models we show that mice immunized with multiple doses of a serotype 8 bioconjugate vaccine elicit antibody responses that mediate opsonophagocytic killing, protect mice from systemic infection, and decrease the ability of serotype 8 pneumococci to colonize the nasopharynx and disseminate. Collectively, these studies demonstrate the utility of bioconjugation to produce efficacious pneumococcal conjugate vaccines.


Assuntos
Infecções Pneumocócicas , Vacinas Pneumocócicas , Animais , Anticorpos Antibacterianos , Proteínas de Transporte , Escherichia coli , Camundongos , Polissacarídeos Bacterianos , Vacinas Conjugadas
17.
ACS Infect Dis ; 7(11): 3111-3123, 2021 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-34633812

RESUMO

Group B Streptococcus (GBS) is a leading cause of neonatal infections and invasive diseases in nonpregnant adults worldwide. Developing a protective conjugate vaccine targeting the capsule of GBS has been pursued for more than 30 years; however, it has yet to yield a licensed product. In this study, we present a novel bioconjugation platform for producing a prototype multivalent GBS conjugate vaccine and its subsequent analytical and immunological characterizations. Using a glycoengineering strategy, we generated strains of Escherichia coli that recombinantly express the type Ia, type Ib, and type III GBS capsular polysaccharides. We then combined the type Ia-, Ib-, and III-capsule-expressing E. coli strains with an engineered Pseudomonas aeruginosa exotoxin A (EPA) carrier protein and the PglS oligosaccharyltransferase. Coexpression of a GBS capsule, the engineered EPA protein, and PglS enabled the covalent attachment of the target GBS capsule to an engineered serine residue on EPA, all within the periplasm of E. coli. GBS bioconjugates were purified, analytically characterized, and evaluated for immunogenicity and functional antibody responses. This proof-of-concept study signifies the first step in the development of a next-generation multivalent GBS bioconjugate vaccine, which was validated by the production of conjugates that are able to elicit functional antibodies directed against the GBS capsule.


Assuntos
Escherichia coli , Infecções Estreptocócicas , Adulto , Anticorpos Antibacterianos , Escherichia coli/genética , Humanos , Recém-Nascido , Infecções Estreptocócicas/prevenção & controle , Streptococcus agalactiae/genética , Vacinas Combinadas
18.
Nat Commun ; 10(1): 891, 2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30792408

RESUMO

Chemical synthesis of conjugate vaccines, consisting of a polysaccharide linked to a protein, can be technically challenging, and in vivo bacterial conjugations (bioconjugations) have emerged as manufacturing alternatives. Bioconjugation relies upon an oligosaccharyltransferase to attach polysaccharides to proteins, but currently employed enzymes are not suitable for the generation of conjugate vaccines when the polysaccharides contain glucose at the reducing end, which is the case for ~75% of Streptococcus pneumoniae capsules. Here, we use an O-linking oligosaccharyltransferase to generate a polyvalent pneumococcal bioconjugate vaccine with polysaccharides containing glucose at their reducing end. In addition, we show that different vaccine carrier proteins can be glycosylated using this system. Pneumococcal bioconjugates are immunogenic, protective and rapidly produced within E. coli using recombinant techniques. These proof-of-principle experiments establish a platform to overcome limitations of other conjugating enzymes enabling the development of bioconjugate vaccines for many important human and animal pathogens.


Assuntos
Escherichia coli/genética , Engenharia Genética/métodos , Vacinas Pneumocócicas/genética , Animais , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/imunologia , Escherichia coli/metabolismo , Glicoproteínas/genética , Glicoproteínas/imunologia , Glicoproteínas/isolamento & purificação , Glicosilação , Humanos , Vacinas Pneumocócicas/isolamento & purificação , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/imunologia , Vacinas Conjugadas/genética , Vacinas Conjugadas/isolamento & purificação , Vacinas Sintéticas/genética , Vacinas Sintéticas/isolamento & purificação
19.
Nat Rev Microbiol ; 16(2): 91-102, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29249812

RESUMO

Acinetobacter baumannii is a nosocomial pathogen that causes ventilator-associated as well as bloodstream infections in critically ill patients, and the spread of multidrug-resistant Acinetobacter strains is cause for concern. Much of the success of A. baumannii can be directly attributed to its plastic genome, which rapidly mutates when faced with adversity and stress. However, fundamental virulence mechanisms beyond canonical drug resistance were recently uncovered that enable A. baumannii and, to a limited extent, other medically relevant Acinetobacter species to successfully thrive in the health-care environment. In this Review, we explore the molecular features that promote environmental persistence, including desiccation resistance, biofilm formation and motility, and we discuss the most recently identified virulence factors, such as secretion systems, surface glycoconjugates and micronutrient acquisition systems that collectively enable these pathogens to successfully infect their hosts.


Assuntos
Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/patogenicidade , Infecções por Acinetobacter/patologia , Acinetobacter baumannii/genética , Acinetobacter baumannii/fisiologia , Infecção Hospitalar/microbiologia , Humanos , Virulência
20.
Trends Microbiol ; 25(7): 532-545, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28216293

RESUMO

Infections caused by the bacterial pathogen Acinetobacter baumannii are a mounting concern for healthcare practitioners as widespread antibiotic resistance continues to limit therapeutic treatment options. The biological processes used by A. baumannii to cause disease are not well defined, but recent research has indicated that secreted proteins may play a major role. A variety of mechanisms have now been shown to contribute to protein secretion by A. baumannii and other pathogenic species of Acinetobacter, including a type II secretion system (T2SS), a type VI secretion system (T6SS), autotransporter, and outer membrane vesicles (OMVs). In this review, we summarize the current knowledge of secretion systems in Acinetobacter species, and highlight their unique aspects that contribute to the pathogenicity and persistence of these emerging pathogens.


Assuntos
Acinetobacter baumannii/metabolismo , Sistemas de Secreção Tipo II , Sistemas de Secreção Tipo V , Sistemas de Secreção Tipo VI , Infecções por Acinetobacter/microbiologia , Infecções por Acinetobacter/fisiopatologia , Acinetobacter baumannii/patogenicidade , Proteínas da Membrana Bacteriana Externa
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