Sputum Proteome Signatures of Mechanically Ventilated Intensive Care Unit Patients Distinguish Samples with or without Anti-pneumococcal Activity

Abstract

Mechanically ventilated patients are at risk of contracting pneumonia. Therefore, these patients often receive prophylactic systemic antimicrobial therapy. Intriguingly however, a previous study showed that antimicrobial activity in bronchoalveolar aspirates (here referred to as “sputa”) from ventilated patients was only partially explained by antibiotic therapy. Here we report that sputa from these patients presented distinct proteome signatures depending on the presence or absence of antimicrobial activity. Moreover, we show that the same distinction applied to antibodies against Streptococcus pneumoniae, which is a major causative agent of pneumonia. Specifically, the investigated sputa that inhibited growth of S. pneumoniae, while containing subinhibitory levels of the antibiotic cefotaxime, presented elevated levels of proteins implicated in innate immune defenses, including complement and apolipoprotein-associated proteins. In contrast, S. pneumoniae-inhibiting sputa with relatively high cefotaxime concentrations or noninhibiting sputa contained higher levels of proteins involved in inflammatory responses, such as neutrophil elastase-associated proteins. In an immunoproteomics analysis, 18 out of 55 S. pneumoniae antigens tested showed significantly increased levels of IgGs in inhibiting sputa. Hence, proteomics and immunoproteomics revealed elevated levels of antimicrobial host proteins or S. pneumoniae antigen-specific IgGs in pneumococcal growth-inhibiting sputa, thus explaining their anti-pneumococcal activity. IMPORTANCE Respiratory pathogens like Streptococcus pneumoniae can cause severe pneumonia. Nonetheless, mechanically ventilated intensive care patients, who have a high risk of contracting pneumonia, rarely develop pneumococcal pneumonia. This suggests the presence of potentially protective antimicrobial agents in their lung environment. Our present study shows for the first time that bronchoalveolar aspirates, “sputa,” of ventilated patients in a Dutch intensive care unit were characterized by three distinct groups of proteome abundance signatures that can explain their anti-pneumococcal activity. Importantly, this anti-pneumococcal sputum activity was related either to elevated levels of antimicrobial host proteins or to antibiotics and S. pneumoniae-specific antibodies. Further, the sputum composition of some patients changed over time. Therefore, we conclude that our study may provide a novel tool to measure changes that are indicative of infection-related conditions in the lungs of mechanically ventilated patients.

Polyvalent Immunoglobulin Preparations Inhibit Pneumolysin-Induced Platelet Destruction

“…The determination of Ply-specific antibody titers in IVIG and human plasma samples was performed via a Luminex-based serological assay described recently. 20 In brief, recombinant pneumolysin was immobilized on xMAP MagPlex beads (Luminex, United States), which were incubated with fourfold serially diluted IVIG (98% IgG; Privigen, CSL Behring, United States) or plasma samples (seven dilutions ranging from 1:50 to 1:204,800). Detection of primary antibody binding was enabled by incubation with an R-Phycoerythrin-labeled anti-human IgG (Jackson ImmunoResearch Europe Ltd, Ely, United Kingdom) followed by measurement in the FLEXMAP 3D (Luminex, United States).…”

The role of proteomics and metabolomics in severe infections

“…A preliminary study found that the BAL fluid from 53% of patients under mechanical ventilation showed antimicrobial activity against three indicator bacteria ( Streptococcus pneumoniae, Staphylococcus aureus, and Streptococcus anginosus ) [24]. In a follow-up study, the authors performed a proteome analysis of the BAL and found that the antipneumococcal activity was associated with higher levels of proteins involved in innate immunity (complement and apolipoprotein-associated) [25 ▪▪ ]. Therefore, these different signatures of proteome and immunoproteome in BAL could explain at least partially differences in the risk of VAP development.…”