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.

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.

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

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