Proteomic Adaptation of Streptococcus pneumoniae to the Human Antimicrobial Peptide LL-37

Secreted antimicrobial peptides (AMPs) are an important part of the human innate immune system and prevent local and systemic infections by inhibiting bacterial growth in a concentration-dependent manner. In the respiratory tract, the cationic peptide LL-37 is one of the most abundant AMPs and capable of building pore complexes in usually negatively charged bacterial membranes, leading to the destruction of bacteria. However, the adaptation mechanisms of several pathogens to LL-37 are already described and are known to weaken the antimicrobial effect of the AMP, for instance, by repulsion, export or degradation of the peptide. This study examines proteome-wide changes in Streptococcus pneumoniae D39, the leading cause of bacterial pneumonia, in response to physiological concentrations of LL-37 by high-resolution mass spectrometry. Our data indicate that pneumococci may use some of the known adaptation mechanisms to reduce the effect of LL-37 on their physiology, too. Additionally, several proteins seem to be involved in resistance to AMPs which have not been related to this process before, such as the teichoic acid flippase TacF (SPD_1128). Understanding colonization- and infection-relevant adaptations of the pneumococcus to AMPs, especially LL-37, could finally uncover new drug targets to weaken the burden of this widespread pathogen

Proteomic adaptation of bacterial pathogens to antimicrobial peptides

Infections with bacterial pathogens are a major cause of morbidity and mortality worldwide. Furthermore, the extensive use of antibiotics increased the frequency of infections with drug-resistant pathogens. Streptococcus pneumoniae, a major cause of bacterial pneumonia, is among the pathogens that often show resistances. As an additional side effect, the use of antibiotics can disrupt the patient’s intestinal microbiome, allowing Clostridioides difficile to cause severe, recurring and hard-to-treat colitis. Hence, new antimicrobials are needed to combat infections caused by these pathogens. A promising approach is the usage of antimicrobial peptides (AMPs), defense molecules produced by organisms from all domains of life. AMPs can specifically perforate bacterial membranes and stimulate the overall immune response of the host. In this work, the proteomic adaptations of S. pneumoniae to the human antimicrobial peptides LL-37 and hBD3 were assessed by high-resolution mass spectrometry and compared to general membrane stress, in order to evaluate the specificity of the bacterial reactions. Furthermore, C. difficile was challenged with the Lactococcus lactis-derived AMP nisin, and the proteomic alterations were examined. In essence, application of LL-37 and hBD3 changed the abundance of pneumococcal proteins involved in membrane transport, including a putative AMP transporter, a protease, virulence proteins and genetic regulators. Moreover, a challenge with LL-37 caused an increase of proteins involved in cell surface modifications that alter the bacterial membrane charge and repel cationic molecules such as LL-37. In support of this, mutants unable to express these proteins were more sensitive to LL-37. In contrast, general membrane stress, induced by the application of cationic detergents, produced a diverse proteomic adjustment, though the same two-component regulatory system was activated. In C. difficile, levels of flagella proteins were significantly increased shortly after treatment with nisin, being in accordance with subsequent electron microscopy data and pointing at a role of these proteins in adaptation to nisin. Interestingly, a flagella-overexpressing mutant showed an enhanced resistance towards nisin, independent of bacterial motility. Taken together, the bacterial pathogens under investigation seem to possess mechanisms to reduce the effect of AMPs on their physiology, a finding that should be considered developing drugs based on AMPs. Although AMPs exhibit membrane perturbations as a common mechanism of action, bacterial adaptation to AMPs appear multifactorial and dependent on the exact pathogen observed and AMP used.Infektionen mit bakteriellen Pathogenen tragen zu einem erheblichen Teil zur weltweiten Morbidität und Mortalität bei. Zusätzlich ist der Anteil an Infekten mit antibiotikaresistenten Krankheitserregern durch den extensiven Einsatz antibakterieller Wirkstoffe gestiegen. Unter den häufig resistenten Pathogenen befindet sich Streptococcus pneumoniae, ein bedeutender Auslöser der bakteriellen Pneumonie. Weiterhin kann eine Behandlung mit Antibiotika die Darmflora des Patienten zerstören und es Clostridioides difficile ermöglichen, eine ernsthafte, wiederkehrende und schwer zu behandelnde Kolitis zu verursachen. Für die Bekämpfung von Infektionen, die diese Pathogene hervorrufen, werden daher neue antimikrobielle Wirkstoffe benötigt. Ein vielversprechender Ansatz ist die Verwendung von antimikrobiellen Peptiden (AMPs), Abwehrmolekülen, die von Organismen aus allen Domänen des Lebens gebildet werden. AMPs können zielgerichtet bakterielle Membranen perforieren und das Immunsystem des Wirtes stimulieren. In dieser Arbeit wurde die Anpassung des Proteoms von S. pneumoniae als Reaktion auf die humanen antimikrobiellen Peptide LL-37 und hBD3 mittels hochauflösender Massenspektrometrie untersucht und zur Beurteilung der Spezifität der Adaptionen mit generellem Membranstress verglichen. Zudem wurde C. difficile dem aus Lactococcus lactis stammenden AMP Nisin ausgesetzt und die dadurch induzierten Veränderungen im Proteom analysiert. Im Wesentlichen hat der Einsatz von LL-37 und hBD3 die Abundanz von Membrantransportproteinen, inklusive eines putativen AMP Transporters, einer Protease, Virulenz-assoziierter Proteine, sowie von genetischen Regulatoren der Pneumokokken beeinflusst. Außerdem führte eine Behandlung mit LL- 37 zu einer Akkumulation von Zellwand-modifizierenden Proteinen, welche die bakterielle Oberflächenladung verändern und dadurch positiv geladene Moleküle wie LL- 37 abstoßen. Zusätzlich zeigten Mutanten, die diese Proteine nicht exprimieren können, eine erhöhte Sensitivität gegenüber LL-37. Im Gegensatz dazu führte Detergensinduzierter genereller Membranstress zu einer abweichenden Proteomanpassung, auch wenn das gleiche regulatorische Zweikomponentensystem aktiviert wurde. In C. difficile war die Menge von Flagellenproteinen kurz nach Zugabe von Nisin signifikant erhöht, was durch elektronenmikroskopische Aufnahmen bestätigt werden konnte und eine Rolle dieser Proteine in der Anpassung an Nisin nahelegt. Interessanterweise wies eine Flagellen-überexprimierende Mutante eine deutliche Resistenz gegenüber Nisin auf, die unabhängig von der bakteriellen Motilität war. Zusammenfassend deuten die Ergebnisse darauf hin, dass die untersuchten bakteriellen Pathogene Mechanismen besitzen, welche die Auswirkungen von AMPs auf ihre Physiologie begrenzen. Dies sollte bei der Entwicklung von Medikamenten basierenden auf AMPs beachtet werden. Obwohl AMPs die Bildung von Membranpertubationen als gemeinsamen Wirkmechanismus aufweisen, sind bakterielle Anpassungsmechanismen offenbar multifaktoriell und hängen von dem untersuchten Pathogen und verwendeten AMP ab

Proteomic Adaptation of Clostridioides difficile to Treatment with the Antimicrobial Peptide Nisin

Clostridioides difficile is the leading cause of antibiotic-associated diarrhea but can also result in more serious, life-threatening conditions. The incidence of C. difficile infections in hospitals is increasing, both in frequency and severity, and antibiotic-resistant C. difficile strains are advancing. Against this background antimicrobial peptides (AMPs) are an interesting alternative to classic antibiotics. Information on the effects of AMPs on C. difficile will not only enhance the knowledge for possible biomedical application but may also provide insights into mechanisms of C. difficile to adapt or counteract AMPs. This study applies state-of-the-art mass spectrometry methods to quantitatively investigate the proteomic response of C. difficile 630∆erm to sublethal concentrations of the AMP nisin allowing to follow the cellular stress adaptation in a time-resolved manner. The results do not only point at a heavy reorganization of the cellular envelope but also resulted in pronounced changes in central cellular processes such as carbohydrate metabolism. Further, the number of flagella per cell was increased during the adaptation process. The potential involvement of flagella in nisin adaptation was supported by a more resistant phenotype exhibited by a non-motile but hyper-flagellated mutant