Streptococcus pneumoniae (Spn) must acquire iron from the host to establish infection. We examined the impact of hemoglobin, the largest iron reservoir in the body, on pneumococcal physiology. Supplementation with hemoglobin allowed Spn to resume growth in an iron-deplete medium. Pneumococcal growth with hemoglobin was unusually robust, exhibiting a prolonged logarithmic growth, higher biomass, and extended viability in both iron-deplete and standard medium. We observed the hemoglobin-dependent response in multiple serotypes, but not with other host proteins, free iron, or heme. Remarkably, hemoglobin induced a sizable transcriptome remodeling, effecting virulence and metabolism in particular genes facilitating host glycoconjugates use. Accordingly, Spn was more adapted to grow on the human α − 1 acid glycoprotein as a sugar source with hemoglobin. A mutant in the hemoglobin/heme-binding protein Spbhp-37 was impaired for growth on heme and hemoglobin iron. The mutant exhibited reduced growth and iron content when grown in THYB and hemoglobin. In summary, the data show that hemoglobin is highly beneficial for Spn cultivation in vitro and suggest that hemoglobin might drive the pathogen adaptation in vivo. The hemoglobin receptor, Spbhp-37, plays a role in mediating the positive influence of hemoglobin. These novel findings provide intriguing insights into pneumococcal interactions with its obligate human host.

The rapid and persistent emergence of drug-resistant bacteria poses a looming public health crisis. The possible task of developing new sets of antibiotics to replenish the existing ones is daunting to say the least. Searching for adjuvants that restore or even enhance the potency of existing antibiotics against drug-resistant strains of bacteria represents a practical and cost-effective approach. Herein, we describe the discovery of potent adjuvants that extend the antimicrobial spectrum of existing antibiotics and restore their effectiveness toward drug-resistant strains including mcr-1-expressing strains. From a library of cationic compounds, MD-100, which has a diamidine core structure, was identified as a potent antibiotic adjuvant against Gram-negative bacteria. Further optimization efforts including the synthesis of ∼20 compounds through medicinal chemistry work led to the discovery of a much more potent compound MD-124. MD-124 was shown to sensitize various Gram-negative bacterial species and strains, including multidrug resistant pathogens, toward existing antibiotics with diverse mechanisms of action. We further demonstrated the efficacy of MD-124 in an ex vivo skin infection model and in an in vivo murine systemic infection model using both wild-type and drug-resistant Escherichia coli strains. MD-124 functions through selective permeabilization of the outer membrane of Gram-negative bacteria. Importantly, bacteria exhibited low-resistance frequency toward MD-124. In-depth computational investigations of MD-124 binding to the bacterial outer membrane using equilibrium and steered molecular dynamics simulations revealed key structural features for favorable interactions. The very potent nature of such adjuvants distinguishes them as very useful leads for future drug development in combating bacterial drug resistance.

Streptococcus pneumoniae grows in biofilms during both asymptomatic colonization and infection. Pneumococcal biofilms on abiotic surfaces exhibit delayed growth and lower biomass and lack the structures seen on epithelial cells or during nasopharyngeal carriage. We show here that adding hemoglobin to the medium activated unusually early and vigorous biofilm growth in multiple S. pneumoniae serotypes grown in batch cultures on abiotic surfaces. Human blood (but not serum, heme, or iron) also stimulated biofilms, and the pore-forming pneumolysin, ply, was required for this induction. S. pneumoniae transitioning from planktonic into sessile growth in the presence of hemoglobin displayed an extensive transcriptome remodeling within 1 and 2 h. Differentially expressed genes included those involved in the metabolism of carbohydrates, nucleotides, amino acid, and lipids. The switch into adherent states also influenced the expression of several regulatory systems, including the comCDE genes. Inactivation of comC resulted in 67% reduction in biofilm formation, while the deletion of comD or comE had limited or no effect, respectively. These observations suggest a novel route for CSP-1 signaling independent of the cognate ComDE two-component system. Biofilm induction and the associated transcriptome remodeling suggest hemoglobin serves as a signal for host colonization in pneumococcus.