Faculty Bibliography

Colonization of the human airways, the first step in the pathogenesis of Streptococcus pneumoniae (Spn), is the determining factor in the ecological spread of the bacterium. Since co-colonization by multiple strains is common, within-host bacterial competition contributes to the success of Spn strains. Competition both between and within strains is mediated by bacteriocin gene clusters, notably the quorum sensing-regulated bacteriocin-like peptide (blp) locus. A key component of this system is the BlpAB transporter that exports pheromones and bacteriocins expressed by the blp locus. However, ~75% of Spn strains lack a functional BlpAB transporter and instead rely on the paralogous ComAB transporter for this export, raising questions about the evolutionary persistence of BlpAB(+) strains. Using molecular barcoding, we demonstrate that BlpAB(+) and BlpAB(-) strains show major differences in population dynamics during colonization modeled in mice. The BlpAB(+) strains exhibit slower loss of clonal diversity as a consequence of intrastrain competition relative to their isogenic BlpAB(-). The contribution of a functional BlpAB transporter was then examined in an association study of >2,000 human carriage isolates from a highly colonized population. The median carriage duration was ~177 days longer for BlpAB(+) relative to BlpAB(-) strains. This increased duration of natural carriage correlates with a competitive advantage for BlpAB(+) strains when tested in the murine model. Thus, our work provides insight into how differences in the population dynamics of Spn mediated by bacterial competition impact host colonization.IMPORTANCESpn is a frequent colonizer of the human upper respiratory tract. Success during colonization is dictated by the arsenal of weapons these bacteria possess, which provides them with an advantage over their competitors. A key example includes the blp bacteriocins that are exported by the cell through both BlpAB and ComAB transporters. While most Spn strains lack a functional BlpAB, a subset of the strains retains it. Given this redundancy in export systems, our study questioned the evolutionary advantage of retaining BlpAB. Herein, we show that a functional BlpAB transporter causes a slower loss of clonal diversity in vivo. This correlates with longer Spn carriage duration in the human population and a competitive advantage during experimental co-colonization. Our work highlights the reasons behind the persistence of Spn with a functional BlpAB. These findings reveal how genetic variability in the blp locus shapes Spn colonization and evolutionary success.

UNLABELLED: IMPORTANCE/OBJECTIVE:research should consider capsule size, not just its presence and type. The results imply that standardized vaccine efficacy tests may yield variable results depending on the capsule production of target strains.

UNLABELLED:(Spn) upregulates neuraminidases (NA) that cleave sialic acid (SA) from host glycans. Because sialylation is thought to contribute to the physical properties that determine mucus function, we posited that Spn directly alters host mucus through NA activity. By directly imaging the colonized URT, we demonstrated NA-mediated alterations to the characteristics and distribution of mucus along the respiratory epithelium, where colonizing bacteria are found. Mucus exposed to NA showed increased localization within goblet cells and lining the glycocalyx. By contrast, NA-naïve mucus was more likely to be observed sloughing away from the epithelial surface. We also visualized Spn in the URT and observed that NA promoted efficient bacterial localization to the firm mucus layer overlying the glycocalyx, whereas NA-deficient Spn was associated more with loose mucus. By facilitating tighter association with the glycocalyx, NA promoted increased Spn colonization density. The magnitude of the NA-mediated effect on colonization was widened during late colonization by increased evasion of host-mediated clearance mechanisms. Thus, Spn-encoded NAs directly modify the host environment by desialylating mucus, which allows close interaction with mucus at the epithelium, and this is associated with enhanced bacterial colonization. IMPORTANCE/OBJECTIVE:Although severe illness and death caused by Spn result from secondary invasive diseases including pneumonia, sepsis, and meningitis, stable colonization of the upper respiratory tract (URT) is a prerequisite to invasive disease. Therefore, understanding host-Spn dynamics during asymptomatic colonization of the URT is warranted with respect to the pathogenesis of Spn disease. In this study, we found that Spn NA activity directly alters mucus characteristics that result in increased density and duration of URT colonization. Therefore, targeting Spn NA activity during URT colonization may be a viable strategy to mitigate Spn infection.

Using chromosomal barcoding, we observed that >97% of the Streptococcus pneumoniae (Spn) population turns over in the lung within 2 days post-inoculation in a murine model. This marked collapse of diversity and bacterial turnover was associated with acute inflammation (severe pneumococcal pneumonia), high bacterial numbers in the lungs, bacteremia, and mortality. Intra-strain competition mediated by the blp locus, which expresses bacteriocins in a quorum-sensing-dependent manner, was required for each of these effects. Bacterial turnover from the activity of Blp-bacteriocins increased the release of the pneumococcal toxin, pneumolysin (Ply), which was sufficient to account for the lung pathology. The ability of Ply to evade complement, rather than its pore-forming activity, prevented opsonophagocytic clearance of Spn enabling its multiplication in the lung, facilitating the inflammatory response and subsequent invasion into the bloodstream. Thus, our study demonstrates how an appreciation for bacterial population dynamics during infection provides new insight into pathogenesis.

Epidemiological studies report the impact of co-infection with pneumococcus and respiratory viruses upon disease rates and outcomes, but their effect on pneumococcal carriage acquisition and bacterial load is scarcely described. Here, we assess this by combining natural viral infection with controlled human pneumococcal infection in 581 healthy adults screened for upper respiratory tract viral infection before intranasal pneumococcal challenge. Across all adults, respiratory syncytial virus (RSV) and rhinovirus asymptomatic infection confer a substantial increase in secondary infection with pneumococcus. RSV also has a major impact on pneumococcal density up to 9 days post challenge. We also study rates and kinetics of bacterial shedding through the nose and oral route in a subset. High levels of pneumococcal colonization density and nasal inflammation are strongly correlated with increased odds of nasal shedding as opposed to cough shedding. Protection against respiratory viral infections and control of pneumococcal density may contribute to preventing pneumococcal disease and reducing bacterial spread.

Atg8 paralogs, consisting of LC3A/B/C and GBRP/GBRPL1/GATE16, function in canonical autophagy; however, their function is controversial because of functional redundancy. In innate immunity, xenophagy and non-canonical single membranous autophagy called "conjugation of Atg8s to single membranes" (CASM) eliminate bacteria in various cells. Previously, we reported that intracellular Streptococcus pneumoniae can induce unique hierarchical autophagy comprised of CASM induction, shedding, and subsequent xenophagy. However, the molecular mechanisms underlying these processes and the biological significance of transient CASM induction remain unknown. Herein, we profile the relationship between Atg8s, autophagy receptors, poly-ubiquitin, and Atg4 paralogs during pneumococcal infection to understand the driving principles of hierarchical autophagy and find that GATE16 and GBRP sequentially play a pivotal role in CASM shedding and subsequent xenophagy induction, respectively, and LC3A and GBRPL1 are involved in CASM/xenophagy induction. Moreover, we reveal ingenious bacterial tactics to gain intracellular survival niches by manipulating CASM-xenophagy progression by generating intracellular pneumococci-derived H₂O₂.

Infants are highly susceptible to invasive respiratory and gastrointestinal infections. To elucidate the age-dependent mechanism(s) that drive bacterial spread from the mucosa, we developed an infant mouse model using the prevalent pediatric respiratory pathogen, Streptococcus pneumoniae (Spn). Despite similar upper respiratory tract (URT) colonization levels, the survival rate of Spn-infected infant mice was significantly decreased compared to adults and corresponded with Spn dissemination to the bloodstream. An increased rate of pneumococcal bacteremia in early life beyond the newborn period was attributed to increased bacterial translocation across the URT barrier. Bacterial dissemination in infant mice was independent of URT monocyte or neutrophil infiltration, phagocyte-derived ROS or RNS, inflammation mediated by toll-like receptor 2 or interleukin 1 receptor signaling, or the pore-forming toxin pneumolysin. Using molecular barcoding of Spn, we found that only a minority of bacterial clones in the nasopharynx disseminated to the blood in infant mice, indicating the absence of robust URT barrier breakdown. Rather, transcriptional profiling of the URT epithelium revealed a failure of infant mice to upregulate genes involved in the tight junction pathway. Expression of many such genes was also decreased in early life in humans. Infant mice also showed increased URT barrier permeability and delayed mucociliary clearance during the first two weeks of life, which corresponded with tighter attachment of bacteria to the respiratory epithelium. Together, these results demonstrate a window of vulnerability during postnatal development when altered mucosal barrier function facilitates bacterial dissemination.

The agr quorum-sensing system links Staphylococcus aureus metabolism to virulence, in part by increasing bacterial survival during exposure to lethal concentrations of H₂O₂, a crucial host defense against S. aureus. We now report that protection by agr surprisingly extends beyond post-exponential growth to the exit from stationary phase when the agr system is no longer turned on. Thus, agr can be considered a constitutive protective factor. Deletion of agr resulted in decreased ATP levels and growth, despite increased rates of respiration or fermentation at appropriate oxygen tensions, suggesting that Δagr cells undergo a shift towards a hyperactive metabolic state in response to diminished metabolic efficiency. As expected from increased respiratory gene expression, reactive oxygen species (ROS) accumulated more in the agr mutant than in wild-type cells, thereby explaining elevated susceptibility of Δagr strains to lethal H₂O₂ doses. Increased survival of wild-type agr cells during H₂O₂ exposure required sodA, which detoxifies superoxide. Additionally, pretreatment of S. aureus with respiration-reducing menadione protected Δagr cells from killing by H₂O₂. Thus, genetic deletion and pharmacologic experiments indicate that agr helps control endogenous ROS, thereby providing resilience against exogenous ROS. The long-lived 'memory' of agr-mediated protection, which is uncoupled from agr activation kinetics, increased hematogenous dissemination to certain tissues during sepsis in ROS-producing, wild-type mice but not ROS-deficient (Cybb

The ongoing transmission of influenza A viruses (IAV) for the past century continues to be a burden to humans. IAV binds terminal sialic acids (SA) of sugar molecules present within the upper respiratory tract (URT) in order to successfully infect hosts. The two most common SA structures that are important for IAV infection are those with α2,3- and α2,6-linkages. While mice were once considered to be an unsuitable system for studying IAV transmission due to their lack of α2,6-SA in the trachea, we have successfully demonstrated that IAV transmission in infant mice is remarkably efficient. This finding led us to re-evaluate the SA composition of the URT of mice using in situ immunofluorescence and examine its in vivo contribution to transmission for the first time. We demonstrate that mice express both α2,3- and α2,6-SA in the URT and that the difference in expression between infants and adults contributes to the variable transmission efficiencies observed. Furthermore, selectively blocking α2,3-SA or α2,6-SA within the URT of infant mice using lectins was necessary but insufficient at inhibiting transmission, and simultaneous blockade of both receptors was crucial in achieving the desired inhibitory effect. By employing a broadly acting neuraminidase to indiscriminately remove both SA moieties in vivo, we effectively suppressed viral shedding and halted the transmission of different strains of influenza viruses. These results emphasize the utility of the infant mouse model for studying IAV transmission and strongly indicate that broadly targeting host SA is an effective approach that inhibits IAV contagion.IMPORTANCEInfluenza virus transmission studies have historically focused on viral mutations that alter hemagglutinin binding to sialic acid (SA) receptors in vitro. However, SA binding preference does not fully account for the complexities of influenza A virus transmission in humans. Our previous findings reveal that viruses that are known to bind α2,6-SA in vitro have different transmission kinetics in vivo, suggesting that diverse SA interactions may occur during their life cycle. In this study, we examine the role of host SA on viral replication, shedding, and transmission in vivo. We highlight the critical role of SA presence during virus shedding, such that attachment to SA during virion egress is equally important as detachment from SA during virion release. These insights support the potential of broadly acting neuraminidases as therapeutic agents capable of restraining viral transmission in vivo. Our study unveils intricate virus-host interactions during shedding, highlighting the necessity to develop innovative strategies to effectively target transmission.