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SUMMARYType VIIb secretion systems (T7SSb) in Gram-positive bacteria facilitate physiology, interbacterial competition, and/or virulence via EssC ATPase-driven secretion of small ɑ-helical proteins and toxins. Recently, we characterized T7SSb in group BStreptococcus(GBS), a leading cause of infection in newborns and immunocompromised adults. GBS T7SS comprises four subtypes based on variation in the C-terminus of EssC and the repertoire of downstream effectors; however, the intra-species diversity of GBS T7SS and impact on GBS-host interactions remains unknown. Bioinformatic analysis indicates that GBS T7SS loci encode subtype-specific putative effectors, which have low inter-species and inter-subtype homology but contain similar domains/motifs and therefore may serve similar functions. We further identify orphaned GBS WXG100 proteins. Functionally, we show that GBS T7SS subtype I and III strains secrete EsxAin vitroand that in subtype I strain CJB111,esxA1appears to be differentially transcribed from the T7SS operon. Further, we observe subtype-specific effects of GBS T7SS on host colonization, as subtype I but not subtype III T7SS promotes GBS vaginal persistence. Finally, we observe that T7SS subtypes I and II are the predominant subtypes in clinical GBS isolates. This study highlights the potential impact of T7SS heterogeneity on host-GBS interactions.

Small, noncoding RNAs (sRNAs) are being increasingly identified as important regulatory molecules in prokaryotes. Due to the prevalence of next-generation sequencing-based techniques, such as RNA sequencing (RNA-seq), there is potential for increased discovery of sRNAs within bacterial genomes; however, these elements are rarely included in annotation files. Consequently, expression values for sRNAs are omitted from most transcriptomic analyses, and mechanistic studies have lagged behind those of protein regulators in numerous bacteria. Two previous studies have identified sRNAs in the human pathogen group B Streptococcus (GBS). Here, we utilize the data from these studies to create updated genome annotation files for the model GBS strains NEM316 and COH1. Using the updated COH1 annotation file, we reanalyze publicly available GBS RNA-seq whole-transcriptome data from GenBank to monitor GBS sRNA expression under a variety of conditions and genetic backgrounds. This analysis generated expression values for 232 putative sRNAs that were overlooked in previous transcriptomic analyses in 21 unique comparisons. To demonstrate the utility of these data, we identify an sRNA that is upregulated during vaginal colonization and demonstrate that overexpression of this sRNA leads to increased bacterial invasion into host epithelial cells. Finally, to monitor RNA degradation, we perform a transcript stability assay to identify highly stable sRNAs and compare stability profiles of sRNA- and protein-coding genes. Collectively, these data provide a wealth of transcriptomic data for putative sRNAs in GBS and a platform for future mechanistic studies. IMPORTANCE In recent years, sRNAs have emerged as potent regulatory molecules in bacteria, including numerous streptococcal species, and contribute to diverse processes, including stress response, metabolism, housekeeping, and virulence regulation. Improvements in sequencing technologies and in silico analyses have facilitated identification of these regulatory molecules as well as improved attempts to determine the location of sRNA genes on the genome. However, despite these advancements, sRNAs are rarely included in genome annotation files. Consequently, these molecules are often omitted from transcriptomic data analyses and are commonly repeat identified across multiple studies. Updating current genomes to include sRNA genes is therefore critical for better understanding bacterial regulation.

The polysaccharide capsule that surrounds Streptococcus pneumoniae (Spn) is one of its most important virulence determinants, serving to protect against phagocytosis. To date, 100 biochemical and antigenically distinct capsule types, i.e., serotypes, of Spn have been identified. Yet how capsule influences pneumococcal translocation across vascular endothelial cells (VEC), a key step in the progression of invasive disease, was unknown. Here, we show that despite capsule being inhibitory of Spn uptake by VEC, capsule enhances the escape rate of internalized pneumococci and thereby promotes translocation. Upon investigation, we determined that capsule protected Spn against intracellular killing by VEC and H2O2-mediated killing in vitro. Using a nitroblue tetrazolium reduction assay and nuclear magnetic resonance (NMR) analyses, purified capsule was confirmed as having antioxidant properties which varied according to serotype. Using an 11-member panel of isogenic capsule-switch mutants, we determined that serotype affected levels of Spn resistance to H2O2-mediated killing in vitro, with killing resistance correlated positively with survival duration within VEC, rate of transcytosis to the basolateral surface, and human attack rates. Experiments with mice supported our in vitro findings, with Spn producing oxidative-stress-resistant type 4 capsule being more organ-invasive than that producing oxidative-stress-sensitive type 2 capsule during bacteremia. Capsule-mediated protection against intracellular killing was also observed for Streptococcus pyogenes and Staphylococcus aureus. We conclude that capsular polysaccharide plays an important role within VEC, serving as an intracellular antioxidant, and that serotype-dependent differences in antioxidant capabilities impact the efficiency of VEC translocation and a serotype's potential for invasive disease. IMPORTANCE Streptococcus pneumoniae (Spn) is the leading cause of invasive disease. Importantly, only a subset of the 100 capsule types carried by Spn cause the majority of serious infections, suggesting that the biochemical properties of capsular polysaccharide are directly tied to virulence. Here, we describe a new function for Spn's capsule-conferring resistance to oxidative stress. Moreover, we demonstrate that capsule promotes intracellular survival of pneumococci within vascular endothelial cells and thereby enhances bacterial translocation across the vasculature and into organs. Using isogenic capsule-switch mutants, we show that different capsule types, i.e., serotypes, vary in their resistance to oxidative stress-mediated killing and that resistance is positively correlated with intracellular survival in an in vitro model, organ invasion during bacteremia in vivo, and epidemiologically established pneumococcal attack rates in humans. Our findings define a new role of capsule and provide an explanation for why certain serotypes of Spn more frequently cause invasive pneumococcal disease.

Group B Streptococcus (GBS) is an asymptomatic colonizer of the female reproductive tract but can cause maternal and neonatal infections and adverse pregnancy outcomes. Here, we closed the genome sequence of strain CJB111, a neonatal GBS clinical isolate from a case of late-onset bacteremia without focus (Houston, TX; 1990).

Group B Streptococcus (GBS) asymptomatically colonizes the female reproductive tract but is a common causative agent of meningitis. GBS meningitis is characterized by extensive infiltration of neutrophils carrying high concentrations of calprotectin, a metal chelator. To persist within inflammatory sites and cause invasive disease, GBS must circumvent host starvation attempts. Here, we identified global requirements for GBS survival during calprotectin challenge, including known and putative systems involved in metal ion transport. We characterized the role of zinc import in tolerating calprotectin stress in vitro and in a mouse model of infection. We observed that a global zinc uptake mutant was less virulent than the parental GBS strain and found calprotectin knockout mice to be equally susceptible to infection by wild-type (WT) and mutant strains. These findings suggest that calprotectin production at the site of infection results in a zinc-limited environment and reveals the importance of GBS metal homeostasis to invasive disease., Nutritional immunity is an elegant host mechanism used to starve invading pathogens of necessary nutrient metals. Calprotectin, a metal-binding protein, is produced abundantly by neutrophils and is found in high concentrations within inflammatory sites during infection. Group B Streptococcus (GBS) colonizes the gastrointestinal and female reproductive tracts and is commonly associated with severe invasive infections in newborns such as pneumonia, sepsis, and meningitis. Although GBS infections induce robust neutrophil recruitment and inflammation, the dynamics of GBS and calprotectin interactions remain unknown. Here, we demonstrate that disease and colonizing isolate strains exhibit susceptibility to metal starvation by calprotectin. We constructed a mariner transposon (Krmit) mutant library in GBS and identified 258 genes that contribute to surviving calprotectin stress. Nearly 20% of all underrepresented mutants following treatment with calprotectin are predicted metal transporters, including known zinc systems. As calprotectin binds zinc with picomolar affinity, we investigated the contribution of GBS zinc uptake to overcoming calprotectin-imposed starvation. Quantitative reverse transcriptase PCR (qRT-PCR) revealed a significant upregulation of genes encoding zinc-binding proteins, adcA, adcAII, and lmb, following calprotectin exposure, while growth in calprotectin revealed a significant defect for a global zinc acquisition mutant (ΔadcAΔadcAIIΔlmb) compared to growth of the GBS wild-type (WT) strain. Furthermore, mice challenged with the ΔadcAΔadcAIIΔlmb mutant exhibited decreased mortality and significantly reduced bacterial burden in the brain compared to mice infected with WT GBS; this difference was abrogated in calprotectin knockout mice. Collectively, these data suggest that GBS zinc transport machinery is important for combatting zinc chelation by calprotectin and establishing invasive disease.

Enterococcus faecalis is a Gram-positive commensal bacterium native to the gastrointestinal tract and an opportunistic pathogen of increasing clinical concern. E. faecalis also colonizes the female reproductive tract and reports suggest vaginal colonization increases following antibiotic treatment or in patients with aerobic vaginitis. Currently, little is known about specific factors that promote E. faecalis vaginal colonization and subsequent infection. We modified an established mouse vaginal colonization model to explore E. faecalis vaginal carriage and demonstrate that both vancomycin resistant and sensitive strains colonize the murine vaginal tract. Following vaginal colonization, we observed E. faecalis in vaginal, cervical and uterine tissue. A mutant lacking endocarditis- and biofilm-associated pili (Ebp) exhibited a decreased ability to associate with human vaginal and cervical cells in vitro, but did not contribute to colonization in vivo. Thus, we screened a low-complexity transposon (Tn) mutant library to identify novel genes important for E. faecalis colonization and persistence in the vaginal tract. This screen revealed 383 mutants that were underrepresented during vaginal colonization at 1, 5 and 8 days post-inoculation compared to growth in culture medium. We confirmed that mutants deficient in ethanolamine catabolism or in the type VII secretion system were attenuated in persisting during vaginal colonization. These results reveal the complex nature of vaginal colonization and suggest that multiple factors contribute to E. faecalis persistence in the reproductive tract.IMPORTANCEDespite increasing prevalence and association of E. faecalis with aerobic vaginitis, essentially nothing is known about the bacterial factors that influence E. faecalis vaginal colonization. We have adapted an animal model of vaginal colonization that supports colonization of multiple E. faecalis strains. Additionally, we determined that ethanolamine utilization and type VII secretion system genes contribute to vaginal colonization and persistence. Identification of factors important for vaginal colonization and persistence provides potential targets for the development of therapeutics. This study is the first to identify key determinants that promote vaginal colonization by E. faecalis, which may represent an important reservoir for antibiotic resistant enterococci.

Type VII secretion systems (T7SS) have been identified in Actinobacteria and Firmicutes and have been shown to secrete effector proteins with functions in virulence, host toxicity, and/or interbacterial killing in a few genera. Bioinformatic analysis indicates that isolates of Group B Streptococcus (GBS) encode at least four distinct subtypes of T7SS machinery, three of which encode adjacent putative T7SS effectors with WXG and LXG motifs. However, the function of T7SS in GBS pathogenesis is unknown. Here we assessed the role of the most abundant GBS T7SS subtype during GBS pathogenesis. In a murine model of hematogenous meningitis, mice infected with GBS lacking a functional T7SS or lacking the secreted WXG100 effector EsxA exhibited less mortality, lower bacterial burdens in tissues, and decreased inflammation in the brain compared to mice infected with the parental GBS strain. We further showed that this T7SS induces cytotoxicity in brain endothelium and that EsxA contributes to these cytotoxicity phenotypes in a WXG motif-dependent manner. Finally, we determined that EsxA is a pore-forming protein, thus demonstrating the first role for a non-mycobacterial EsxA homolog in pore formation. This work reveals the importance of a T7SS in host–GBS interactions and has implications for T7SS effector function in other Gram-positive bacteria., Author summary Group B Streptococcus (GBS) is an important human pathogen that is a leading cause of invasive disease in newborns and certain adult populations, including pregnant women, the elderly, and those with diabetes. During pregnancy, asymptomatically colonizing GBS in the female genital tract can be transmitted to the fetus or newborn and can result in neonatal meningitis upon GBS disruption of the blood-brain barrier (BBB). GBS encodes a type VII secretion system (T7SS), which may allow export of proteins and/or toxins that promote BBB disruption; however, the GBS T7SS has not been studied. Here we show that GBS encodes four types of T7SSs and that the most prevalent subtype is important for GBS meningitis progression, possibly by inducing inflammation and cell death in the brain. We also show that a secreted T7SS effector protein, EsxA, contributes to GBS pathogenesis and can form pores in lipid membranes. This is the first demonstration of EsxA-mediated pore-formation in Gram-positive bacteria.

Group B Streptococcus (GBS) is a major opportunistic pathogen in certain adult populations, including pregnant women, and remains a leading etiologic agent of newborn disease. During pregnancy, GBS asymptomatically colonizes the vaginal tract of 20–30% of healthy women, but can be transmitted to the neonate in utero or during birth resulting in neonatal pneumonia, sepsis, meningitis, and subsequently 10–15% mortality regardless of antibiotic treatment. While various GBS virulence factors have been implicated in vaginal colonization and invasive disease, the regulation of many of these factors remains unclear. Recently, CRISPR-associated protein-9 (Cas9), an endonuclease known for its role in CRISPR/Cas immunity, has also been observed to modulate virulence in a number of bacterial pathogens. However, the role of Cas9 in GBS colonization and disease pathogenesis has not been well-studied. We performed allelic replacement of cas9 in GBS human clinical isolates of the hypervirulent sequence-type 17 strain lineage to generate isogenic Δcas9 mutants. Compared to parental strains, Δcas9 mutants were attenuated in murine models of hematogenous meningitis and vaginal colonization and exhibited significantly decreased invasion of human brain endothelium and adherence to vaginal epithelium. To determine if Cas9 alters transcription in GBS, we performed RNA-Seq analysis and found that 353 genes (>17% of the GBS genome) were differentially expressed between the parental WT and Δcas9 mutant strain. Significantly dysregulated genes included those encoding predicted virulence factors, metabolic factors, two-component systems (TCS), and factors important for cell wall formation. These findings were confirmed by qRT-PCR and suggest that Cas9 may regulate a significant portion of the GBS genome. We studied one of the TCS regulators, CiaR, that was significantly downregulated in the Δcas9 mutant strain. RNA-Seq analysis of the WT and ΔciaR strains demonstrated that almost all CiaR-regulated genes were also significantly regulated by Cas9, suggesting that Cas9 may modulate GBS gene expression through other regulators. Further we show that CiaR contributes to GBS vaginal colonization and persistence. Altogether, these data highlight the potential complexity and importance of the non-canonical function of Cas9 in GBS colonization and disease.

Among 98 serotypes of Streptococcus pneumoniae, only a small subset regularly causes invasive pneumococcal diseases (IPD). We previously demonstrated that serotype 11A binds to ficolin-2 and has low invasiveness in children. Epidemiologic data suggested, however, that serotype 11A IPD afflicts older adults, possibly indicating reduced ficolin-2-mediated immune protection. Therefore, we studied the epidemiology of ficolin-2-bound serotypes. We obtained IPD case data from the United States Centers for Disease Control and Prevention. We studied three prominent ficolin-2-bound serotypes and their acetyltransferase-deficient variants for ficolin-2 binding and ficolin-2-mediated complement deposition with flow-cytometry. We determined the age distributions of these serotypes from the obtained epidemiologic data. We discovered that the serotype 35B capsule is a novel ficolin-2 ligand due to O-acetylation via WciG. Ficolin-2-mediated complement deposition was observed on serotypes 11A and 35B but not serotype 31 or any O-acetyl transferase deficient derivatives of these serotypes. Serotypes 11A, 35B, and 31 cause more IPD among older adults than children. Studies of the three serotypes provide additional evidence for ficolin-2 providing innate immunity against IPD. The skewed age distribution of the three serotypes suggests that older adults have reduced ficolin-2-mediated immunity and are more susceptible to these serotypes.

Streptococcus agalactiae (group B Streptococcus [GBS]) is often a commensal bacterium that colonizes healthy adults asymptomatically and is a frequent inhabitant of the vaginal tract in women. However, in immunocompromised individuals, particularly the newborn, GBS may transition to an invasive pathogen and cause serious disease. Despite the use of the currently recommended intrapartum antibiotic prophylaxis for GBS-positive mothers, GBS remains a leading cause of neonatal septicemia and meningitis. To adapt to the various host environments encountered during its disease cycle, GBS possesses multiple two-component regulatory systems (TCSs). Here we investigated the contribution of a transcriptional regulator containing a LytTR domain, LtdR, to GBS pathogenesis. Disruption of the ltdR gene in the GBS chromosome resulted in a significant increase in bacterial invasion into human cerebral microvascular endothelial cells (hCMEC) in vitro as well as the greater penetration of the blood-brain barrier (BBB) and the development of meningitis in vivo . Correspondingly, infection of hCMEC with the Δ ltdR mutant resulted in increased secretion of the proinflammatory cytokines interleukin-8 (IL-8), CXCL-1, and IL-6. Further, using a mouse model of GBS vaginal colonization, we observed that the Δ ltdR mutant was cleared more readily from the vaginal tract and also that infection with the Δ ltdR mutant resulted in increased cytokine production from human vaginal epithelial cells. RNA sequencing revealed global transcriptional differences between the Δ ltdR mutant and the parental wild-type GBS strain. These results suggest that LtdR regulates many bacterial processes that can influence GBS-host interactions to promote both bacterial persistence and disease progression.

As a species, Streptococcus pneumoniae (the pneumococcus) utilizes a diverse array of capsular polysaccharides to evade the host. In contrast to large variations in sugar composition and linkage formation, O-acetylation is a subtle capsular modification that nonetheless has a large impact on capsular shielding and recognition of the capsule by vaccine-elicited antibodies. Serotype 15B, which is included in the 23-valent pneumococcal polysaccharide vaccine (PPV23), carries the putative O-acetyltransferase gene wciZ . The coding sequence of wciZ contains eight consecutive TA repeats [(TA) 8 ]. Replication slippage is thought to result in the addition or loss of TA repeats, subsequently causing frameshift and truncation of WciZ to yield a nonacetylated serotype, 15C. Using sensitive serological tools, we show that serotype 15C isolates whose wciZ contains seven or nine TA repeats retain partial O-acetylation, while serotype 15C isolates whose wciZ contains six TA repeats have barely detectable O-acetylation. We confirmed by inhibition enzyme-linked immunosorbent assay that (TA) 7 serotype 15C is ∼0.1% as acetylated as serotype 15B, while serotype 15X is nonacetylated. To eliminate the impact of genetic background, we created isogenic serotype 15B, (TA) 7 serotype 15C, and 15BΔ wciZ (15X) strains and found that reduction or absence of WciZ-mediated O-acetylation did not affect capsular shielding from phagocytes, biofilm formation, adhesion to nasopharyngeal cells, desiccation tolerance, or murine colonization. Sera from PPV23-immunized persons opsonized serotype 15B significantly but only slightly better than serotypes 15C and 15X; thus, PPV23 may not result in expansion of serotype 15C.

Streptococcus pneumoniae (pneumococcus) produces many capsule types that differ in their abilities to evade host immune recognition. To explain these serotype-dependent protective capacities, many studies have investigated capsular thickness or the interaction of the capsule with complement proteins, but the effects of small chemical modifications of the capsule on its function have not been studied. One small chemical modification found frequently among pneumococcal capsules is O-acetylation. Pneumococcal serotype 33A has two membrane-bound O -acetyltransferase genes, wciG and wcjE . A 33A wcjE -deficient variant, 33F, occurs naturally and is increasing in prevalence in the wake of widespread conjugate vaccine use, but no wciG -deficient variants have been reported. To study the biological consequence of the loss of O-acetylation, we created wciG -deficient variants in both serotypes 33A and 33F, which we named 33X1 (Δ wciG ) and 33X2 (Δ wciG Δ wcjE ). Serotypes 33X1 and 33X2 express novel capsule types based on serological and biochemical analyses. We found that loss of WcjE-mediated O-acetylation appears not to affect cell wall shielding, since serotypes 33A and 33F exhibit comparable nonspecific opsonophagocytic killing, biofilm production, and adhesion to nasopharyngeal cells, though serotype 33F survived short-term drying better than serotype 33A. Loss of WciG-mediated O-acetylation in serotypes 33X1 and 33X2, however, resulted in a phenotype resembling that of nonencapsulated strains: increased cell wall accessibility, increased nonspecific opsonophagocytic killing, enhanced biofilm formation, and increased adhesion to nasopharyngeal cells. We conclude that WciG-mediated, but not WcjE-mediated, O-acetylation is important for producing protective capsules in 33A and that small chemical changes to the capsule can drastically affect its biological properties.

Streptococcus agalactiae, also known as Group B Streptococcus (GBS), is a Gram-positive bacterium isolated from the vaginal tract of approximately 25% of women. GBS colonization of the female reproductive tract is of particular concern during pregnancy as the bacteria can invade gestational tissues or be transmitted to the newborn during passage through the birth canal. Infection of the neonate can result in life-threatening pneumonia, sepsis and meningitis. Thus, surveillance of GBS strains and corresponding virulence potential during colonization is warranted. Here we describe a panel of GBS isolates from the vaginal tracts of a cohort of pregnant women in Michigan, USA. We determined that capsular serotypes III and V were the most abundant across the strain panel, with only one isolate belonging to serotype IV. Further, 12.8% of strains belonged to the hyper-virulent serotype III, sequence type 17 (ST-17) and 15.4% expressed the serine rich repeat glycoprotein-encoding gene srr2. Functional assessment of the colonizing isolates revealed that almost all strains exhibited some level of β-hemolytic activity and that ST-17 strains, which express Srr2, exhibited increased bacterial adherence to vaginal epithelium. Finally, analysis of strain antibiotic susceptibility revealed the presence of antibiotic resistance to penicillin (15.4%), clindamycin (30.8%), erythromycin (43.6%), vancomycin (30.8%), and tetracycline (94.9%), which has significant implications for treatment options. Collectively, these data provide important information on vaginal GBS carriage isolate virulence potential and highlight the value of continued surveillance.

The human opsonin ficolin-2 (L-ficolin) is an innate pattern-recognizing molecule that binds to acetylated moieties. Upon binding, ficolin-2 activates complement through the lectin pathway, opsonizing the target to promote phagocytic clearance. Ficolin-2 has been found to interact with a growing number of pathogenic bacteria, fungi, and viruses. Ficolin-2 also has proposed roles in host homeostasis, including the clearance of apoptotic cells. Consequently, there is an increased interest in studying ficolin-2, and access to purified ficolin-2 is necessary for these studies. Ficolin-2 purified from serum, plasma, or cell culture supernatants has been a useful tool in the characterization of ficolin-2 function; however, available protocols are laborious and inefficient, requiring additional processing of starting materials (e.g., polyethylene glycol precipitation or dialysis) and multiple steps of purification. Here, we investigated a simple solution to the problem: use of a simple, disposable bioreactor requiring only standard tissue culture equipment. Using this system, we generated cell culture supernatants containing high concentrations of recombinant ficolin-2, which permitted rapid purification of high-purity recombinant ficolin-2 without processing the supernatants. Purified recombinant ficolin-2 retained its binding capacity and supported complement activation in vitro. Bioreactor cultivation will likely be generally useful in the production of other recombinant proteins in the study of the complement system.

BACKGROUND: Recent studies have identified Streptococcus pneumoniae serotype 11E and serovariant 11Av among isolates previously typed as 11A by classical serotyping methods. Serotype 11E and serovariant 11Av differ from serotype 11A by having totally or partially inactive wcjE, a gene in cps locus coding for an O-acetyl transferase. Serotype 11E is rare among carriage isolates but common among invasive isolates suggesting that it survives better during invasion. Aim of this work was to investigate the epidemiology of serotype 11A in a pneumococcal collection using a new serotyping approach based on High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance (HR-MAS NMR) spectroscopy to distinguish serotypes 11A and 11E. METHODS: A collection of 48 (34 invasive and 14 carriage) S. pneumoniae isolates from Italy, previously identified as serotype 11A by the Quellung reaction, were investigated by wcjE sequencing, HR-MAS NMR spectroscopy and the reference flow cytometric serotyping assay (FCSA) based on monoclonal antibodies. RESULTS: HR-MAS NMR spectra from serotypes 11A and 11E showed different NMR peaks indicating that HR-MAS NMR could be used to distinguish these serotypes, although HR-MAS NMR could not distinguish serotype 11Av from serotype 11E unambiguously. Thirty-eight isolates were confirmed to be serotype 11A, 8 isolates with a mutated wcjE were serotype 11E, 1 isolate belonged to serovariant 11Av, and 1 isolate was a mixed population 11A/11Av. All 11E isolates were identified among invasive isolates. CONCLUSIONS: We proved that HR-MAS NMR can be of potential use for pneumococcal serotyping. The detection of serotype 11E among invasive isolates in our collection, supports previous epidemiological studies suggesting that mutations in wcjE can represent a mechanism promoting pneumococcal survival during invasion. The discovery of a spectrum of immunochemical diversity within established serotypes should stimulate efforts to develop new serotyping approaches.

The ficolins are members of a recently discovered family of host innate opsonins that can activate the lectin pathway of complement. The ficolins bind many ligands, although they are typically described as binding acetylated sugars. Ficolin-1 (M-ficolin) and ficolin-2 (L-ficolin) are known to bindStreptococcus pneumoniaeserotypes 19C and 11A, respectively. While studying the binding of ficolins to pneumococci, we found variations in ficolin-2 binding among serum samples collected in different types of blood collection tubes. Plastic tubes, which contain a silica clot activator, yielded sera with reduced ficolin-2 binding and apparent ficolin-2 levels. We found that the silica clot activator eluted from plastic red-top tubes inhibited ficolin-2 ligand binding, while other related proteins, like mannose-binding lectin (MBL) and ficolin-1, were not affected. These tube types did not affect the concentrations of other related opsonins (C1q, MBL, or ficolin-3 [H-ficolin]). Interestingly, we also found that ficolin-1 levels were increased 2- to 3-fold in plastic serum separator tubes compared to the increases in other tube types. These findings have implications for future ficolin-1 and ficolin-2 studies, as proper sample collection and handling are essential.