In a recently published study, Dr American Journal of Physiology-Lung Cellular and Molecular PhysiologyA group of researchers examined how heparan sulfate (HS) shedding affects the efficacy of cathelicidin in methicillin-resistant Staphylococcus aureus (MRSA) pneumonia.
Pneumonia, especially caused by MRSA, is a leading cause of infectious death. The mechanisms leading to staphylococcal pneumonia are not fully understood. This study investigates the interactions between MRSA, the pulmonary epithelial glycocalyx and antimicrobial peptides (AMP) in pneumonia.
The focus is on the HS-rich glycocalyx, a sulfated layer lining the alveoli that is known to bind cationic proteins. We examined the shedding of HS in the airways after lung injury and its possible effects on lung function and interactions with AMPs. Specifically, we investigate how HS shed oligosaccharides, specifically during bacterial pneumonia, interact with cathelicidin-like AMPs, influencing host immune responses and pathogen dynamics.
Further research is needed to fully understand the mechanisms by which HS shedding affects AMP function, providing potential for novel therapeutic strategies in the treatment of pneumonia.
About the study
In this study, following University of Colorado Institutional Animal Care and Use Committee (IACUC) and Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines, male C57BL6 mice underwent intratracheal MRSA instillation, followed by bronchoalveolar lavage (Fluidlana). . Researchers used mass spectrometry to measure HS in BAL fluid, blinded to treatment group for objective outcomes.
Additionally, the team innovatively collected airway fluid from pneumonia patients using heat and moisture exchanger (HME) filters at Vanderbilt University Medical Center under an approved Institutional Review Board (IRB) protocol. It aims to detect lung changes due to respiratory failure.
The study employed surface plasmon resonance (SPR) to examine the binding dynamics between AMP and HS, providing insight into real-time, label-free interactions. Simultaneously, bacterial growth curves under different conditions were studied to evaluate the effect of different heparin types on MRSA strains.
A detailed process of bacterial ribonucleic acid (RNA) isolation and sequencing was conducted, involving MRSA culture in heparin or saline, followed by RNA extraction and sequencing. These steps were important for exploring transcriptomic changes and understanding bacterial pneumonia dynamics.
The study also included quantification of minimum inhibitory concentration (MIC) for different pneumonia pathogens against AMP at different HS concentrations. This was important in assessing how HS affects AMP efficacy against bacterial infection. Rigorous statistical analysis confirmed that the study results were reliable and valid.
Results of the study
In the current study, the researchers used a murine model of MRSA pneumonia. Mass spectrometry analysis revealed a significant increase in HS in the airway lining fluid of MRSA-infected mice compared with saline controls. Notably, this increase was characterized by a higher abundance of sulfated HS, especially multi-sulfated disaccharides. Complementary analyzes of HME filter samples indicated higher HS levels in patients with Gram-negative pneumonia than in patients with Gram-positive pneumonia, suggesting a tenuous relationship between bacterial etiology and HS shedding.
Despite the observed increase in shed HS in the lung environment, the study did not find a direct effect of HS on MRSA growth or gene transcription. Experiments involving different sizes and sulfation patterns of HS showed no significant changes in MRSA growth or transcriptomic response. This finding suggested that HS, although an important component of lung status after injury, does not directly inhibit bacterial growth or alter bacterial gene expression.
The study further elucidated the interaction between HS and host immune mediators. Using surface plasmon resonance (SPR), the researchers quantified the binding of HS to murine cathelicidin-related antimicrobial peptide (mCRAMP). The observed strong binding indicates a possible interaction in vivo, which could potentially affect the host response to bacterial infection.
Most critically, the study investigated the functional implications of HS binding to mCRAMP. Focusing on common nosocomial pneumonia pathogens including MRSA, Klebsiella pneumoniaeAnd Pseudomonas aeruginosa, The study used a modified radial diffusion assay to evaluate the MIC of mCRAMP against these bacteria.
The results showed a significant increase in MIC with high HS concentrations, indicating a reduced bactericidal effect of mCRAMP in the presence of HS. This discovery was particularly noteworthy because it highlighted the complex interplay between HS and the host defense system, where HS, despite not directly affecting MRSA growth, significantly altered the efficacy of an antimicrobial peptide.
Overall, the study emphasizes the complex dynamics within the pulmonary environment following bacterial pneumonia. Acute shedding of epithelial HS, especially when enriched in the sulfated form, presents a subtle challenge to the host’s immune response, potentially affecting the effectiveness of innate immune mechanisms against bacterial pathogens.