Animal models of Klebsiella pneumoniae mucosal infections.

Klebsiella pneumoniae is among the most relevant pathogens worldwide, causing high morbidity and mortality, which is worsened by the increasing rates of antibiotic resistance. It is a constituent of the host microbiota of different mucosa, that can invade and cause infections in many different sites. The development of new treatments and prophylaxis against this pathogen rely on animal models to identify potential targets and evaluate the efficacy and possible side effects of therapeutic agents or vaccines. However, the validity of data generated is highly dependable on choosing models that can adequately reproduce the hallmarks of human diseases. The present review summarizes the current knowledge on animal models used to investigate K. pneumoniae infections, with a focus on mucosal sites. The advantages and limitations of each model are discussed and compared; the applications, extrapolations to human subjects and future modifications that can improve the current techniques are also presented. While mice are the most widely used species in K. pneumoniae animal studies, they present limitations such as the natural resistance to the pathogen and difficulties in reproducing the main steps of human mucosal infections. Other models, such as Drosophila melanogaster (fruit fly), Caenorhabditis elegans, Galleria mellonella and Danio rerio (zebrafish), contribute to understanding specific aspects of the infection process, such as bacterial lethality and colonization and innate immune system response, however, they but do not present the immunological complexity of mammals. In conclusion, the choice of the animal model of K. pneumoniae infection will depend mainly on the questions being addressed by the study, while a better understanding of the interplay between bacterial virulence factors and animal host responses will provide a deeper comprehension of the disease process and aid in the development of effective preventive/therapeutic strategies.

Robust Immune Response and Protection against Lethal Pneumococcal Challenge with a Recombinant BCG-PspA-PdT Prime/Boost Scheme Administered to Neonatal Mice.

Pneumococcal diseases are an important public health problem, with high mortality rates in young children. Although conjugated pneumococcal vaccines offer high protection against invasive pneumococcal diseases, this is restricted to vaccine serotypes, leading to serotype replacement. Furthermore, the current vaccines do not protect neonates. Therefore, several protein-based pneumococcal vaccines have been studied over the last few decades. Our group established a recombinant BCG expressing rPspA-PdT as a prime/rPspA-PdT boost strategy, which protected adult mice against lethal intranasal pneumococcal challenge. Here, we immunized groups of neonate C57/Bl6 mice (6-10) (at 5 days) with rBCG PspA-PdT and a boost with rPspA-PdT (at 12 days). Controls were saline or each antigen alone. The prime/boost strategy promoted an IgG1 to IgG2c isotype shift compared to protein alone. Furthermore, there was an increase in specific memory cells (T and B lymphocytes) and higher cytokine production (IFN-γ, IL-17, TNF-α, IL-10, and IL-6). Immunization with rBCG PspA-PdT/rPspA-PdT showed 100% protection against pulmonary challenge with the WU2 pneumococcal strain; two doses of rPspA-PdT showed non-significant protection in the neonates. These results demonstrate that a prime/boost strategy using rBCG PspA-PdT/rPspA-PdT is effective in protecting neonates against lethal pneumococcal infection via the induction of strong antibody and cytokine responses.

Robust immune response and protection against lethal pneumococcal challenge with a recombinant BCG-PspA-PdT prime/boost scheme administered to neonatal mice

Pneumococcal diseases are an important public health problem, with high mortality rates in young children. Although conjugated pneumococcal vaccines offer high protection against invasive pneumococcal diseases, this is restricted to vaccine serotypes, leading to serotype replacement. Furthermore, the current vaccines do not protect neonates. Therefore, several protein-based pneumococcal vaccines have been studied over the last few decades. Our group established a recombinant BCG expressing rPspA-PdT as a prime/rPspA-PdT boost strategy, which protected adult mice against lethal intranasal pneumococcal challenge. Here, we immunized groups of neonate C57/Bl6 mice (6–10) (at 5 days) with rBCG PspA-PdT and a boost with rPspA-PdT (at 12 days). Controls were saline or each antigen alone. The prime/boost strategy promoted an IgG1 to IgG2c isotype shift compared to protein alone. Furthermore, there was an increase in specific memory cells (T and B lymphocytes) and higher cytokine production (IFN-γ, IL-17, TNF-α, IL-10, and IL-6). Immunization with rBCG PspA-PdT/rPspA-PdT showed 100% protection against pulmonary challenge with the WU2 pneumococcal strain; two doses of rPspA-PdT showed non-significant protection in the neonates. These results demonstrate that a prime/boost strategy using rBCG PspA-PdT/rPspA-PdT is effective in protecting neonates against lethal pneumococcal infection via the induction of strong antibody and cytokine responses.

Fusion of PspA to detoxified pneumolysin enhances pneumococcal vaccine coverage.

Despite the implementation of conjugate vaccines in several countries, S. pneumoniae continues to pose a great burden worldwide, causing around 1 million annual deaths. Pneumococcal proteins have long been investigated as serotype-independent vaccines against this pathogen, with promising results. However, it is a consensus that one antigen alone will not be sufficient to provide long-term protection with wide coverage. Amongst the most well studied pneumococcal proteins are PspA and pneumolysin (Ply), two major virulence factors required by the bacterium for successful invasion of host tissues. PspA is highly immunogenic and protective, but it is structurally variable; pneumolysin is conserved among different pneumococci, but it is toxic to the host. To overcome these limitations, N-terminal PspA fragments have been genetically fused to non-toxic pneumolysin derivatives (PlD) to create PspA_PlD chimeras. Mouse immunization with these fusions confers protection against pneumococcal strains expressing heterologous PspAs, which correlates with antibody-induced complement C3 deposition on the surface of multiple pneumococcal strains. Analysis of mutant strains lacking PspA or Pneumolysin shows that both proteins contribute to the antibody-mediated enhancement in complement deposition induced by the fusion. These results expand previous data evaluating PspA_PlD and demonstrate that the fusion combines the protective traits of both proteins, inducing antibodies that efficiently promote complement deposition on multiple strains and cross-protection.

Fusion of PspA to detoxified pneumolysin enhances pneumococcal vaccine coverage

Despite the implementation of conjugate vaccines in several countries, S. pneumoniae continues to pose a great burden worldwide, causing around 1 million annual deaths. Pneumococcal proteins have long been investigated as serotype-independent vaccines against this pathogen, with promising results. However, it is a consensus that one antigen alone will not be sufficient to provide long-term protection with wide coverage. Amongst the most well studied pneumococcal proteins are PspA and pneumolysin (Ply), two major virulence factors required by the bacterium for successful invasion of host tissues. PspA is highly immunogenic and protective, but it is structurally variable; pneumolysin is conserved among different pneumococci, but it is toxic to the host. To overcome these limitations, N-terminal PspA fragments have been genetically fused to non-toxic pneumolysin derivatives (PlD) to create PspA_PlD chimeras. Mouse immunization with these fusions confers protection against pneumococcal strains expressing heterologous PspAs, which correlates with antibody-induced complement C3 deposition on the surface of multiple pneumococcal strains. Analysis of mutant strains lacking PspA or Pneumolysin shows that both proteins contribute to the antibody-mediated enhancement in complement deposition induced by the fusion. These results expand previous data evaluating PspA_PlD and demonstrate that the fusion combines the protective traits of both proteins, inducing antibodies that efficiently promote complement deposition on multiple strains and cross-protection.

A fusion protein comprising pneumococcal surface protein A and a pneumolysin derivate confers protection in a murine model of pneumococcal pneumonia.

PspA and pneumolysin are two important vaccine candidates, able to elicit protection in different models of pneumococcal infection. The high immunogenic potential of PspA, combined with a possible adjuvant effect of pneumolysin derivatives (due to their ability to interact with TLR-4) could greatly improve the immunogenicity and coverage of a protein-based pneumococcal vaccine. A chimeric protein including the N-terminal region of PspA in fusion with the pneumolysin derivative, PlD1, has been shown to induce high antibody levels against each protein, and protect mice against invasive challenge. The aim of the present study was to investigate the cellular response induced by such vaccine, and to evaluate protection in a murine model of lobar pneumococcal pneumonia. Pneumococcal pneumonia was induced in BALB/c mice by nasal instillation of a high dose of a serotype 14 strain with low virulence. Airway inflammation was confirmed by total and differential cell counts in BAL and by histological analysis of the lungs, and bacterial loads were measured 7 days after challenge. Cytokine levels were determined in the bronchoalveolar fluid (BALF) of mice immunized with rPspA-PlD1 fusion after challenge, by flow cytometry and ELISA. After challenge, the mice developed lung inflammation with no invasion of other sites, as demonstrated by histological analysis. We detected significant production of TNF-α and IL-6 in the BALF, which correlated with protection against pneumonia in the group immunized with rPspA-PlD1. In conclusion, we found that the rPspA-PlD1fusion is protective against pneumococcal pneumonia in mice, and protection is correlated with an early and controlled local inflammatory response. These results are in agreement with previous data demonstrating the efficacy of the fusion protein against pneumococcal sepsis and reinforce the potential of the rPspA-PlD1 protein chimera as a promising vaccine strategy to prevent pneumococcal disease.

Host-microbiome interactions regarding peri-implantitis and dental implant loss.

In the last decades, the ortho-aesthetic-functional rehabilitation had significant advances with the advent of implantology. Despite the success in implantology surgeries, there is a percentage of failures mainly due to in loco infections, through bacterial proliferation, presence of fungi and biofilm formation, originating peri-implantitis. In this sense, several studies have been conducted since then, seeking answers to numerous questions that remain unknown. Thus, the present work aims to discuss the interaction between host-oral microbiome and the development of peri-implantitis. Peri-implantitis was associated with a diversity of bacterial species, being Porphiromonas gingivalis, Treponema denticola and Tannerella forsythia described in higher proportion of peri-implantitis samples. In a parallel role, the injury of peri-implant tissue causes an inflammatory response mediated by activation of innate immune cells such as macrophages, dendritic cells, mast cells, and neutrophils. In summary, the host immune system activation may lead to imbalance of oral microbiota, and, in turn, the oral microbiota dysbiosis is reported leading to cytokines, chemokines, prostaglandins, and proteolytic enzymes production. These biological processes may be responsible for implant loss.

Immune response induced in mice by a hybrid rPotD-PdT pneumococcal protein.

Streptococcus pneumoniae is a human pathogen that colonizes the naso and/or oropharynx and can cause otitis, pneumonia, bacteremia and meningitis. To broaden the protection against pneumococcus, several pneumococcal proteins have been investigated as vaccine candidates. In this study we analyzed the immunological response induced by mouse subcutaneous immunization with a fusion of the Polyamine transport protein D (PotD) and a pneumolysin derivative (PdT), resulting in a hybrid rPotD-PdT protein. Immunization of mice with rPotD-PdT induced increased production of nitric oxide, indicating a higher innate immune response. In agreement, immunization of mice with the hybrid protein was more immunogenic than the individual proteins or their combination, eliciting higher antibody levels. The anti-rPotD-PdT IgG displayed increased binding onto the pneumococcal surface. Furthermore, the anti-rPotD-PdT antisera promoted superior opsonophagocytosis as compared with the other tested formulations. However, despite that the encouraging results in vitro, immunization with the hybrid was not sufficient to induce protection against sepsis with a highly virulent pneumococcal strain. taken together, the results suggest that hybrid proteins are an interesting strategy, able to promote improved immune responses, but the inclusion of other antigens may be necessary to promote protection against invasive infections caused by this bacterium.

Klebsiella pneumoniae Biofilms and Their Role in Disease Pathogenesis.

The ability to form biofilms is a crucial virulence trait for several microorganisms, including Klebsiella pneumoniae - a Gram-negative encapsulated bacterium often associated with nosocomial infections. It is estimated that 65-80% of bacterial infections are biofilm related. Biofilms are complex bacterial communities composed of one or more species encased in an extracellular matrix made of proteins, carbohydrates and genetic material derived from the bacteria themselves as well as from the host. Bacteria in the biofilm are shielded from immune responses and antibiotics. The present review discusses the characteristics of K. pneumoniae biofilms, factors affecting biofilm development, and their contribution to infections. We also explore different model systems designed to study biofilm formation in this species. A great number of factors contribute to biofilm establishment and maintenance in K. pneumoniae, which highlights the importance of this mechanism for the bacterial fitness. Some of these molecules could be used in future vaccines against this bacterium. However, there is still a lack of in vivo models to evaluate the contribution of biofilm development to disease pathogenesis. With that in mind, the combination of different methodologies has great potential to provide a more detailed scenario that more accurately reflects the steps and progression of natural infection.

Influence of the Polysaccharide Capsule on the Bactericidal Activity of Indolicidin on Streptococcus pneumoniae.

Streptococcus pneumoniae is a pathogen responsible for high morbidity and mortality worldwide. The polysaccharide capsule confers protection against phagocytosis and influences many aspects of pneumococcal pathogenesis. The capsular polysaccharides (CPS) are highly immunogenic and exhibit great structural variability, with more than 100 serotypes described so far. Antimicrobial peptides (AMPs) are an important part of the innate defense mechanisms against many pathogens. Indolicidin is a cationic AMP produced by bovine neutrophils, with bactericidal effects against several bacteria. CPS has been shown to interfere with the ability of AMPs to kill pneumococci, but the effects of capsule variability on susceptibility to indolicidin have not been explored. The present work determined the effects of capsule on resistance to indolicidin in vitro. Using a bactericidal plate assay, we observed that different pneumococcal serotypes exhibited variable resistance to indolicidin, which correlated with the capsule net charge. Interestingly, the effect of capsule expression on resistance to indolicidin was dependent on the serotype; bacteria with lower zeta potential were more resistant to indolicidin when capsule was present, while those with less negative surface charge were more resistant in the absence of capsule. The addition of purified CPS partially rescued the bacteria from the bactericidal effects of indolicidin, while the addition of anticapsular antibodies accentuated the peptide's bactericidal action, suggesting a possible new protective mechanism induced by polysaccharide-based pneumococcal vaccines.

Association between asthma and enamel defects in primary and young permanent teeth - A systematic review.

Childhood respiratory diseases, such as asthma, are important public health problems worldwide and could be associated with tooth enamel defects. This study aimed to verify the relationship between asthma and enamel defects in teeth, to answer the following question: "Could asthma in children be significantly associated with enamel defects in deciduous dentition and young permanent teeth?." PUBMED-MEDLINE, EMBASE, LILACS, and COCHRANE databases were systematically searched and assessed articles (2000-2021) were cautiously scored according to a predetermined criterion. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses were considered. Twenty-two articles were critically appraised and used as a basis for conclusions. The relationship between asthma and enamel defects was confirmed in the majority of appraised papers, one with a high level of evidence, nine with a moderate level, and four with a low level. Out of the eight manuscripts investigating the influence of asthma medication on enamel defects, only three (one with high, one moderate, and another with a low level of evidence) suggested an association. It can be concluded that asthma is closely connected with enamel defects in young permanent teeth. However, as most of the papers appraised were of cross-sectional or case-control design, further well-designed clinical investigations with a prospective design are welcome to reinforce our findings.

A fusion protein comprising pneumococcal surface protein A and a pneumolysin derivate confers protection in a murine model of pneumococcal pneumonia

PspA and pneumolysin are two important vaccine candidates, able to elicit protection in different models of pneumococcal infection. The high immunogenic potential of PspA, combined with a possible adjuvant effect of pneumolysin derivatives (due to their ability to interact with TLR-4) could greatly improve the immunogenicity and coverage of a proteinbased pneumococcal vaccine. A chimeric protein including the N-terminal region of PspA in fusion with the pneumolysin derivative, PlD1, has been shown to induce high antibody levels against each protein, and protect mice against invasive challenge. The aim of the present study was to investigate the cellular response induced by such vaccine, and to evaluate protection in a murine model of lobar pneumococcal pneumonia. Pneumococcal pneumonia was induced in BALB/c mice by nasal instillation of a high dose of a serotype 14 strain with low virulence. Airway inflammation was confirmed by total and differential cell counts in BAL and by histological analysis of the lungs, and bacterial loads were measured 7 days after challenge. Cytokine levels were determined in the bronchoalveolar fluid (BALF) of mice immunized with rPspA-PlD1 fusion after challenge, by flow cytometry and ELISA. After challenge, the mice developed lung inflammation with no invasion of other sites, as demonstrated by histological analysis. We detected significant production of TNF-α and IL-6 in the BALF, which correlated with protection against pneumonia in the group immunized with rPspA-PlD1. In conclusion, we found that the rPspA-PlD1fusion is protective against pneumococcal pneumonia in mice, and protection is correlated with an early and controlled local inflammatory response. These results are in agreement with previous data demonstrating the efficacy of the fusion protein against pneumococcal sepsis and reinforce the potential of the rPspA-PlD1 protein chimera as a promising vaccine strategy to prevent pneumococcal disease.

Influence of the Polysaccharide capsule on the bactericidal activity of indolicidin on Streptococcus pneumoniae

Streptococcus pneumoniae is a pathogen responsible for high morbidity and mortality worldwide. The polysaccharide capsule confers protection against phagocytosis and influences many aspects of pneumococcal pathogenesis. The capsular polysaccharides (CPS) are highly immunogenic and exhibit great structural variability, with more than 100 serotypes described so far. Antimicrobial peptides (AMPs) are an important part of the innate defense mechanisms against many pathogens. Indolicidin is a cationic AMP produced by bovine neutrophils, with bactericidal effects against several bacteria. CPS has been shown to interfere with the ability of AMPs to kill pneumococci, but the effects of capsule variability on susceptibility to indolicidin have not been explored. The present work determined the effects of capsule on resistance to indolicidin in vitro. Using a bactericidal plate assay, we observed that different pneumococcal serotypes exhibited variable resistance to indolicidin, which correlated with the capsule net charge. Interestingly, the effect of capsule expression on resistance to indolicidin was dependent on the serotype; bacteria with lower zeta potential were more resistant to indolicidin when capsule was present, while those with less negative surface charge were more resistant in the absence of capsule. The addition of purified CPS partially rescued the bacteria from the bactericidal effects of indolicidin, while the addition of anticapsular antibodies accentuated the peptide’s bactericidal action, suggesting a possible new protective mechanism induced by polysaccharide-based pneumococcal vaccines.

Current Stage in the Development of Klebsiella pneumoniae Vaccines.

Klebsiella pneumoniae is a bacterium capable of colonizing mucous membranes, causing serious infections. Widespread antibiotic resistance in K. pneumoniae-either through intrinsic mechanisms or via acquisition from different species, especially in hospital environments-limits the therapeutic options against this pathogen, further aggravating the disease burden. To date, there are no vaccines available against K. pneumoniae infection. Although formulations based on capsular polysaccharides have been proposed, the high variability in capsular serotypes limits vaccine coverage. Recombinant vaccines based on surface exposed bacterial antigens are a promising alternative owing to their conservation among different serotypes and accessibility to the immune system. Many vaccine candidates have been proposed, some of which have reached clinical trials. The present review summarizes the current status of K. pneumoniae vaccine development. Different strategies including whole cell vaccines, outer membrane vesicles (OMVs), ribosome, polysaccharide, lipopolysaccharide (LPS), and protein-based formulations are discussed. The contribution of antibody and cell-mediated responses is also presented. In summary, K. pneumoniae vaccines are feasible and a promising strategy to prevent infections and to reduce the antimicrobial resistance burden worldwide.

SdiA, a Quorum-Sensing Regulator, Suppresses Fimbriae Expression, Biofilm Formation, and Quorum-Sensing Signaling Molecules Production in Klebsiella pneumoniae.

Klebsiella pneumoniae is a Gram-negative pathogen that has become a worldwide concern due to the emergence of multidrug-resistant isolates responsible for various invasive infectious diseases. Biofilm formation constitutes a major virulence factor for K. pneumoniae and relies on the expression of fimbrial adhesins and aggregation of bacterial cells on biotic or abiotic surfaces in a coordinated manner. During biofilm aggregation, bacterial cells communicate with each other through inter- or intra-species interactions mediated by signallng molecules, called autoinducers, in a mechanism known as quorum sensing (QS). In most Gram-negative bacteria, intra-species communication typically involves the LuxI/LuxR system: LuxI synthase produces N-acyl homoserine lactones (AHLs) as autoinducers and the LuxR transcription factor is their cognate receptor. However, K. pneumoniae does not produce AHL but encodes SdiA, an orphan LuxR-type receptor that responds to exogenous AHL molecules produced by other bacterial species. While SdiA regulates several cellular processes and the expression of virulence factors in many pathogens, the role of this regulator in K. pneumoniae remains unknown. In this study, we describe the characterization of sdiA mutant strain of K. pneumoniae. The sdiA mutant strain has increased biofilm formation, which correlates with the increased expression of type 1 fimbriae, thus revealing a repressive role of SdiA in fimbriae expression and bacterial cell adherence and aggregation. On the other hand, SdiA acts as a transcriptional activator of cell division machinery assembly in the septum, since cells lacking SdiA regulator exhibited a filamentary shape rather than the typical rod shape. We also show that K. pneumoniae cells lacking SdiA regulator present constant production of QS autoinducers at maximum levels, suggesting a putative role for SdiA in the regulation of AI-2 production. Taken together, our results demonstrate that SdiA regulates cell division and the expression of virulence factors such as fimbriae expression, biofilm formation, and production of QS autoinducers in K. pneumoniae.

Protective role of PhtD and its amino and carboxyl fragments against pneumococcal sepsis.

The implementation of polysaccharide-based vaccines has massively reduced the incidence of invasive pneumococcal diseases. However, there is great concern regarding serotype replacement and the increase in antibiotic resistant strains expressing non-vaccine capsular types. In addition, conjugate vaccines have high production costs, a limiting factor for their implementation in mass immunization programs in developing countries. These limitations have prompted the development of novel vaccine strategies for prevention of Streptococcus pneumoniae infections. The use of conserved pneumococcal antigens such as recombinant proteins or protein fragments presents an interesting serotype-independent alternative. Pht is a family of surface-exposed proteins which have been evaluated as potential vaccine candidates with encouraging results. The present work investigated the immune responses elicited by subcutaneous immunization of mice with the polyhistidine triad protein D (PhtD) and its amino and carboxyl terminal fragments. The proteins were immunogenic and protective against pneumococcal sepsis in mice. Antibodies raised against PhtD increased complement C3b deposition on the pneumococcal surface, mainly mediated by the alternative pathway. Sera from mice immunized with PhtD and PhtD_Cter promoted an increase in bacterial uptake by mouse phagocytes. The interaction of PhtD with the complement system regulator factor H was investigated in silico and in vitro by ELISA and western blot, confirming PhtD as a factor-H binding protein. Our results support the inclusion of PhtD and more specifically, its C-terminal fragment in a multicomponent serotype independent vaccine and suggests a role for the complement system in PhtD-mediated protection.

Resistance Mechanisms to Antimicrobial Peptides in Gram-Positive Bacteria.

With the alarming increase of infections caused by pathogenic multidrug-resistant bacteria over the last decades, antimicrobial peptides (AMPs) have been investigated as a potential treatment for those infections, directly through their lytic effect or indirectly, due to their ability to modulate the immune system. There are still concerns regarding the use of such molecules in the treatment of infections, such as cell toxicity and host factors that lead to peptide inhibition. To overcome these limitations, different approaches like peptide modification to reduce toxicity and peptide combinations to improve therapeutic efficacy are being tested. Human defense peptides consist of an important part of the innate immune system, against a myriad of potential aggressors, which have in turn developed different ways to overcome the AMPs microbicidal activities. Since the antimicrobial activity of AMPs vary between Gram-positive and Gram-negative species, so do the bacterial resistance arsenal. This review discusses the mechanisms exploited by Gram-positive bacteria to circumvent killing by antimicrobial peptides. Specifically, the most clinically relevant genera, Streptococcus spp., Staphylococcus spp., Enterococcus spp. and Gram-positive bacilli, have been explored.

Immunization with PhtD truncated fragments reduces nasopharyngeal colonization by Streptococcus pneumoniae.

Despite the undeniable success of polysaccharide vaccines against Streptococcus pneumoniae infections, there is a consensus on the scientific field that this approach should be revised in order to overpass the problems related with these formulations, such as serotype replacement and high production costs. The study of conserved pneumococcal proteins or its truncated fragments has emerged as a serotype independent alternative. In this work, we have characterized the immune response elicited by systemic immunization of mice with the Histidine triad protein D (PhtD) and its' amino and carboxyl terminal fragments. The proteins were shown to be immunogenic and protective against pneumococcal colonization, with increased IL-17 production, and induction of antibodies able to limit pneumococcal adhesion to human respiratory cells. Antiserum against PhtD_Nter, but not C_ter or PhtD, promoted an increase in bacterial phagocytosis in vitro. Interestingly, antibodies against the PhtD_Nter displayed cross-reactivity with two other pneumococcal proteins, PspA and PspC, due to sequence similarities in the proline rich region of the molecules. On a whole, our results support the inclusion of PhtD, and more specifically, its N-terminal fragment, in a multicomponent serotype independent vaccine.

Functional Insights From KpfR, a New Transcriptional Regulator of Fimbrial Expression That Is Crucial for Klebsiella pneumoniae Pathogenicity.

Although originally known as an opportunistic pathogen, Klebsiella pneumoniae has been considered a worldwide health threat nowadays due to the emergence of hypervirulent and antibiotic-resistant strains capable of causing severe infections not only on immunocompromised patients but also on healthy individuals. Fimbriae is an essential virulence factor for K. pneumoniae, especially in urinary tract infections (UTIs), because it allows the pathogen to adhere and invade urothelial cells and to form biofilms on biotic and abiotic surfaces. The importance of fimbriae for K. pneumoniae pathogenicity is highlighted by the large number of fimbrial gene clusters on the bacterium genome, which requires a coordinated and finely adjusted system to control the synthesis of these structures. In this work, we describe KpfR as a new transcriptional repressor of fimbrial expression in K. pneumoniae and discuss its role in the bacterium pathogenicity. K. pneumoniae with disrupted kpfR gene exhibited a hyperfimbriated phenotype with enhanced biofilm formation and greater adhesion to and replication within epithelial host cells. Nonetheless, the mutant strain was attenuated for colonization of the bladder in a murine model of urinary tract infection. These results indicate that KpfR is an important transcriptional repressor that, by negatively controlling the expression of fimbriae, prevents K. pneumoniae from having a hyperfimbriated phenotype and from being recognized and eliminated by the host immune system.

Immunization with PhtD truncated fragments reduces nasopharyngeal colonization by Streptococcus pneumoniae

Despite the undeniable success of polysaccharide vaccines against Streptococcus pneumoniae infections, there is a consensus on the scientific field that this approach should be revised in order to overpass the problems related with these formulations, such as serotype replacement and high production costs. The study of conserved pneumococcal proteins or its truncated fragments has emerged as a serotype independent alternative. In this work, we have characterized the immune response elicited by systemic immunization of mice with the Histidine triad protein D (PhtD) and its’ amino and carboxyl terminal fragments. The proteins were shown to be immunogenic and protective against pneumococcal colonization, with increased IL-17 production, and induction of antibodies able to limit pneumococcal adhesion to human respiratory cells. Antiserum against PhtD_Nter, but not C_ter or PhtD, promoted an increase in bacterial phagocytosis in vitro. Interestingly, antibodies against the PhtD_Nter displayed cross-reactivity with two other pneumococcal proteins, PspA and PspC, due to sequence similarities in the proline rich region of the molecules. On a whole, our results support the inclusion of PhtD, and more specifically, its N-terminal fragment, in a multicomponent serotype independent vaccine