The human milk protein-lipid complex HAMLET disrupts glycolysis and induces death in Streptococcus pneumoniae.

HAMLET is a complex of human α-lactalbumin (ALA) and oleic acid and kills several Gram-positive bacteria by a mechanism that bears resemblance to apoptosis in eukaryotic cells. To identify HAMLET's bacterial targets, here we used Streptococcus pneumoniae as a model organism and employed a proteomic approach that identified several potential candidates. Two of these targets were the glycolytic enzymes fructose bisphosphate aldolase (FBPA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Treatment of pneumococci with HAMLET immediately inhibited their ATP and lactate production, suggesting that HAMLET inhibits glycolysis. This observation was supported by experiments with recombinant bacterial enzymes, along with biochemical and bacterial viability assays, indicating that HAMLET's activity is partially inhibited by high glucose-mediated stimulation of glycolysis but enhanced in the presence of the glycolysis inhibitor 2-deoxyglucose. Both HAMLET and ALA bound directly to each glycolytic enzyme in solution and solid-phase assays and effectively inhibited their enzymatic activities. In contrast, oleic acid alone had little to no inhibitory activity. However, ALA alone also exhibited no bactericidal activity and did not block glycolysis in whole cells, suggesting a role for the lipid moiety in the internalization of HAMLET into the bacterial cells to reach its target(s). This was verified by inhibition of enzyme activity in whole cells after HAMLET but not ALA exposure. The results of this study suggest that part of HAMLET's antibacterial activity relates to its ability to target and inhibit glycolytic enzymes, providing an example of a natural antimicrobial agent that specifically targets glycolysis.

Novel Strategy To Protect against Influenza Virus-Induced Pneumococcal Disease without Interfering with Commensal Colonization.

Streptococcus pneumoniae commonly inhabits the nasopharynx as a member of the commensal biofilm. Infection with respiratory viruses, such as influenza A virus, induces commensal S. pneumoniae to disseminate beyond the nasopharynx and to elicit severe infections of the middle ears, lungs, and blood that are associated with high rates of morbidity and mortality. Current preventive strategies, including the polysaccharide conjugate vaccines, aim to eliminate asymptomatic carriage with vaccine-type pneumococci. However, this has resulted in serotype replacement with, so far, less fit pneumococcal strains, which has changed the nasopharyngeal flora, opening the niche for entry of other virulent pathogens (e.g., Streptococcus pyogenes, Staphylococcus aureus, and potentially Haemophilus influenzae). The long-term effects of these changes are unknown. Here, we present an attractive, alternative preventive approach where we subvert virus-induced pneumococcal disease without interfering with commensal colonization, thus specifically targeting disease-causing organisms. In that regard, pneumococcal surface protein A (PspA), a major surface protein of pneumococci, is a promising vaccine target. Intradermal (i.d.) immunization of mice with recombinant PspA in combination with LT-IIb(T13I), a novel i.d. adjuvant of the type II heat-labile enterotoxin family, elicited strong systemic PspA-specific IgG responses without inducing mucosal anti-PspA IgA responses. This response protected mice from otitis media, pneumonia, and septicemia and averted the cytokine storm associated with septic infection but had no effect on asymptomatic colonization. Our results firmly demonstrated that this immunization strategy against virally induced pneumococcal disease can be conferred without disturbing the desirable preexisting commensal colonization of the nasopharynx.

Dynamic changes in the Streptococcus pneumoniae transcriptome during transition from biofilm formation to invasive disease upon influenza A virus infection.

Streptococcus pneumoniae is a leading cause of infectious disease globally. Nasopharyngeal colonization occurs in biofilms and precedes infection. Prior studies have indicated that biofilm-derived pneumococci are avirulent. However, influenza A virus (IAV) infection releases virulent pneumococci from biofilms in vitro and in vivo. Triggers of dispersal include IAV-induced changes in the nasopharynx, such as increased temperature (fever) and extracellular ATP (tissue damage). We used whole-transcriptome shotgun sequencing (RNA-seq) to compare the S. pneumoniae transcriptome in biofilms, bacteria dispersed from biofilms after exposure to IAV, febrile-range temperature, or ATP, and planktonic cells grown at 37°C. Compared with biofilm bacteria, actively dispersed S. pneumoniae, which were more virulent in invasive disease, upregulated genes involved in carbohydrate metabolism. Enzymatic assays for ATP and lactate production confirmed that dispersed pneumococci exhibited increased metabolism compared to those in biofilms. Dispersed pneumococci also upregulated genes associated with production of bacteriocins and downregulated colonization-associated genes related to competence, fratricide, and the transparent colony phenotype. IAV had the largest impact on the pneumococcal transcriptome. Similar transcriptional differences were also observed when actively dispersed bacteria were compared with avirulent planktonic bacteria. Our data demonstrate complex changes in the pneumococcal transcriptome in response to IAV-induced changes in the environment. Our data suggest that disease is caused by pneumococci that are primed to move to tissue sites with altered nutrient availability and to protect themselves from the nasopharyngeal microflora and host immune response. These data help explain pneumococcal virulence after IAV infection and have important implications for studies of S. pneumoniae pathogenesis.

Monitoring changes in membrane polarity, membrane integrity, and intracellular ion concentrations in Streptococcus pneumoniae using fluorescent dyes.

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca(2+), K(+), and H(+), respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Oleic acid is a key cytotoxic component of HAMLET-like complexes.

HAMLET is a complex of α-lactalbumin (α-LA) with oleic acid (OA) that selectively kills tumor cells and Streptococcus pneumoniae. To assess the contribution of the proteinaceous component to cytotoxicity of HAMLET, OA complexes with proteins structurally and functionally distinct from α-LA were prepared. Similar to HAMLET, the OA complexes with bovine ß-lactoglobulin (bLG) and pike parvalbumin (pPA) (bLG-OA-45 and pPA-OA-45, respectively) induced S. pneumoniae D39 cell death. The activation mechanisms of S. pneumoniae death for these complexes were analogous to those for HAMLET, and the cytotoxicity of the complexes increased with OA content in the preparations. The half-maximal inhibitory concentration for HEp-2 cells linearly decreased with rise in OA content in the preparations, and OA concentration in the preparations causing HEp-2 cell death was close to the cytotoxicity of OA alone. Hence, the cytotoxic action of these complexes against HEp-2 cells is induced mostly by OA. Thermal stabilization of bLG upon association with OA implies that cytotoxicity of bLG-OA-45 complex cannot be ascribed to molten globule-like conformation of the protein component. Overall, the proteinaceous component of HAMLET-like complexes studied is not a prerequisite for their activity; the cytotoxicity of these complexes is mostly due to the action of OA.

Role of dihydrolipoamide dehydrogenase in regulation of raffinose transport in Streptococcus pneumoniae.

Streptococcus pneumoniae strains lacking the enzyme dihydrolipoamide dehydrogenase (DLDH) show markedly reduced ability to grow on raffinose and stachyose as sole carbon sources. Import of these sugars occurs through the previously characterized raffinose ATP-binding cassette (ABC) transport system, encoded by the raf operon, that lacks the necessary ATP-binding protein. In this study, we identified the raffinose ATP-binding protein RafK and showed that it was directly involved in raffinose and stachyose import. RafK carries a C-terminal regulatory domain present in a subset of ATP-binding proteins that has been involved in both direct regulation of transporter activity (inducer exclusion) and transcription of transporter genes. Pneumococci lacking RafK showed a 50- to 80-fold reduction in expression of the raf operon genes aga (alpha-galactosidase) and rafEFG (raffinose substrate binding and permease genes), and both glucose and sucrose inhibited raffinose uptake through inducer exclusion. Like RafK, the presence of DLDH also activated the expression of raf operon genes, as DLDH-negative pneumococci showed a significantly decreased expression of aga and rafEFG, but DLDH did not regulate rafK or the putative regulatory genes rafR and rafS. DLDH also bound directly to RafK both in vitro and in vivo, indicating the possibility that DLDH regulates raffinose transport by a direct interaction with the regulatory domain of the transporter. Finally, although not as attenuated as DLDH-negative bacteria, pneumococci lacking RafK were significantly outcompeted by wild-type bacteria in colonization experiments of murine lung and nasopharynx, indicating a role for raffinose and stachyose transport in vivo.

Apoptosis-like death in bacteria induced by HAMLET, a human milk lipid-protein complex.

BACKGROUND: Apoptosis is the primary means for eliminating unwanted cells in multicellular organisms in order to preserve tissue homeostasis and function. It is characterized by distinct changes in the morphology of the dying cell that are orchestrated by a series of discrete biochemical events. Although there is evidence of primitive forms of programmed cell death also in prokaryotes, no information is available to suggest that prokaryotic death displays mechanistic similarities to the highly regulated programmed death of eukaryotic cells. In this study we compared the characteristics of tumor and bacterial cell death induced by HAMLET, a human milk complex of alpha-lactalbumin and oleic acid. METHODOLOGY/PRINCIPAL FINDINGS: We show that HAMLET-treated bacteria undergo cell death with mechanistic and morphologic similarities to apoptotic death of tumor cells. In Jurkat cells and Streptococcus pneumoniae death was accompanied by apoptosis-like morphology such as cell shrinkage, DNA condensation, and DNA degradation into high molecular weight fragments of similar sizes, detected by field inverse gel electrophoresis. HAMLET was internalized into tumor cells and associated with mitochondria, causing a rapid depolarization of the mitochondrial membrane and bound to and induced depolarization of the pneumococcal membrane with similar kinetic and magnitude as in mitochondria. Membrane depolarization in both systems required calcium transport, and both tumor cells and bacteria were found to require serine protease activity (but not caspase activity) to execute cell death. CONCLUSIONS/SIGNIFICANCE: Our results suggest that many of the morphological changes and biochemical responses associated with apoptosis are present in prokaryotes. Identifying the mechanisms of bacterial cell death has the potential to reveal novel targets for future antimicrobial therapy and to further our understanding of core activation mechanisms of cell death in eukaryote cells.

Expression of a uniquely regulated extracellular polysaccharide confers a large-capsule phenotype to Bacteroides fragilis.

Bacteroides fragilis synthesizes eight distinct capsular polysaccharides, more than any described bacterium outside the order Bacteroidales. Here, we show that this organism also produces a high-molecular-weight extracellular polysaccharide (EPS). Expression of the EPS results in the formation of a large polysaccharide layer around the bacteria which prevents them from forming a tight pellet upon centrifugation and from entering a Percoll density gradient. Like expression of the capsular polysaccharides, expression of the EPS is phase variable and dictated by DNA inversion of its promoter. EPS expression is regulated at one level by the DNA invertase Tsr19, which is encoded by a gene immediately upstream of the EPS locus and inverts the EPS promoter, causing an on or off phenotype. Expression of the EPS is also regulated at another level, which dictates the amount of EPS produced. By analyzing a panel of tsr19 deletion mutants, we found that the number of inverted repeats (IRs) flanking the promoter is variable. Transcription into the EPS genes is greater in mutants with a single IR between the promoter and the downstream EPS genes than in mutants with more than one IR in this region, correlating with the synthesis of more EPS. By analyzing the relative orientations of the EPS promoter of bacteria obtained from human fecal samples, we showed that both DNA inversion and variation in the number of IRs are active processes of B. fragilis in the endogenous human intestinal ecosystem.

Bacteroides fragilis synthesizes a DNA invertase affecting both a local and a distant region.

The activity of a fourth conserved tyrosine site-specific recombinase (Tsr) of Bacteroides fragilis was characterized. Its gene, tsr19, is adjacent to mpi, encoding the global DNA invertase regulating capsular polysaccharide biosynthesis. Unlike the other described Tsrs of B. fragilis, Tsr19 brings about inversion of two DNA regions, one local and one located distantly.