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Staphylococcus aureus is a human commensal organism and opportunist pathogen, causing potentially fatal disease. The presence of non-pathogenic microflora or their components, at the point of infection, dramatically increases S. aureus pathogenicity, a process termed augmentation. Augmentation is associated with macrophage interaction but by a hitherto unknown mechanism. Here, we demonstrate a breadth of cross-kingdom microorganisms can augment S. aureus disease and that pathogenesis of Enterococcus faecalis can also be augmented. Co-administration of augmenting material also forms an efficacious vaccine model for S. aureus. In vitro, augmenting material protects S. aureus directly from reactive oxygen species (ROS), which correlates with in vivo studies where augmentation restores full virulence to the ROS-susceptible, attenuated mutant katA ahpC. At the cellular level, augmentation increases bacterial survival within macrophages via amelioration of ROS, leading to proliferation and escape. We have defined the molecular basis for augmentation that represents an important aspect of the initiation of infection., Author summary S. aureus is a commensal inhabitant of the human skin and nares. However, it can cause serious diseases if it is able to breach our protective barriers such as the skin, often via wounds or surgery. If infection occurs via a wound, this initial inoculum contains both the pathogen, other members of the microflora and also wider environmental microbes. We have previously described “augmentation”, whereby this other non-pathogenic material can enhance the ability of S. aureus to lead to a serious disease outcome. Here we have determined the breadth of augmenting material and elucidated the cellular and molecular basis for its activity. Augmentation occurs via shielding of S. aureus from the direct bactericidal effects of reactive oxygen species produced by macrophages. This initial protection enables the effective establishment of S. aureus infection. Understanding augmentation not only explains an important facet of the interaction of S. aureus with our innate immune system, but also provides a platform for the development of novel prophylaxis approaches.

Serum bactericidal assays (SBA) are valuable for assessing the functional activity of natural and vaccine-induced antibodies against many Gram-negative bacteria, such as meningococcus and Salmonella. However, SBA often require an exogenous source of complement and the presence of pre-existing naturally acquired antibodies limits the use of human complement for this purpose. To remove pre-existing Salmonella-specific antibodies, in the context of SBA for Salmonella vaccine research, we incubated human sera with preparations of Salmonella. By incubating at 4 °C, pre-existing antibodies were adsorbed onto the Salmonella bacteria with only minimal complement deposition. We assessed the effects of adsorption on specific antibody levels, complement activity and the bactericidal activity of sera using flow cytometry, SBA and haemolytic assays. Adsorption removed Salmonella-specific antibodies and bactericidal activity against Salmonella from whole serum but was not detrimental to serum complement activity, even after five adsorption cycles. Bactericidal activity could be reconstituted in the adsorbed serum by the addition of exogenous specific antibodies. Sera preadsorbed with Salmonella are suitable as a source of human complement to measure the bactericidal activity of Salmonella antibodies. The adsorption method can be used to deplete, simply and rapidly, specific antibodies from serum to prepare a source of human complement for use in SBA for vaccine research and assessment.

Unassisted metastasis through the lymphatic system is a mechanism of dissemination thus far ascribed only to cancer cells. Here, we report that Streptococcus pyogenes also hijack lymphatic vessels to escape a local infection site, transiting through sequential lymph nodes and efferent lymphatic vessels to enter the bloodstream. Contrasting with previously reported mechanisms of intracellular pathogen carriage by phagocytes, we show S. pyogenes remain extracellular during transit, first in afferent and then efferent lymphatics that carry the bacteria through successive draining lymph nodes. We identify streptococcal virulence mechanisms important for bacterial lymphatic dissemination and show that metastatic streptococci within infected lymph nodes resist and subvert clearance by phagocytes, enabling replication that can seed intense bloodstream infection. The findings establish the lymphatic system as both a survival niche and conduit to the bloodstream for S. pyogenes, explaining the phenomenon of occult bacteraemia. This work provides new perspectives in streptococcal pathogenesis with implications for immunity., Pathogenic agents can spread from an initial to a secondary site via the lymphatics. Here, using a mouse model of infection, the authors show that S. pyogenes readily transit through sequential lymph nodes within efferent lymphatics to reach the bloodstream and drive systemic infection, while remaining extracellular.

Even in nonpandemic times, respiratory viruses account for a vast global burden of disease. They remain a major cause of illness and death and they pose a perpetual threat of breaking out into epidemics and pandemics. Many of these respiratory viruses infect repeatedly and appear to induce only narrow transient immunity, but the situation varies from one virus to another. In the absence of effective specific treatments, understanding the role of immunity in protection, disease, and resolution is of paramount importance. These problems have been brought into sharp focus by the coronavirus disease 2019 (COVID-19) pandemic. Here, we summarise what is now known about adaptive immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and draw comparisons with immunity to other respiratory viruses, focusing on the longevity of protective responses.

Lymphatic vessels carry fluid from tissues back to the bloodstream through filtering lymph nodes that limit infection and restrict passage of pathogens; ability to exploit these conduits is ascribed only to cancer cells and a small number of, principally intracellular, pathogens. Here, we report that Streptococcus pyogenes and other extracellular bacteria rapidly escape a local infection site and enter the blood circulation via the lymphatics, passing through sequential draining lymph nodes, and transiting in inter-nodal efferent lymphatic vessels, not blood vessels. Notably, S. pyogenes remain extracellular within lymphatic vessels and lymph nodes. By subverting innate immune responses to infection foci in lymph nodes, metastatic S. pyogenes replicate and seed intense bacteremia and systemic disease. Our findings identify extracellular lymphatic metastasis as the primary route of bacterial dissemination that provides both a survival niche and a conduit to reach the bloodstream and distant tissues for a range of bacteria.

Invasive group A Streptococcus (iGAS) is frequently associated with emm1 isolates, with an attendant mortality of around 20%. Cases occasionally arise in previously healthy individuals with a history of upper respiratory tract infection, soft tissue contusion, and no obvious portal of entry. Using a new murine model of contusion, we determined the impact of contusion on iGAS bacterial burden and phenotype.\ud \ud Calibrated mild blunt contusion did not provide a focus for initiation or seeding of GAS that was detectable following systemic GAS bacteremia, but instead enhanced GAS migration to the local draining lymph node following GAS inoculation at the same time and site of contusion. Increased migration to lymph node was associated with emergence of mucoid bacteria, although was not specific to mucoid bacteria. In one study, mucoid colonies demonstrated a significant increase in capsular hyaluronan that was not linked to a covRS or rocA mutation, but to a deletion in the promoter of the capsule synthesis locus, hasABC, resulting in a strain with increased fitness for lymph node migration.\ud \ud In summary, in the mild contusion model used, we could not detect seeding of muscle by GAS. Contusion promoted bacterial transit to the local lymph node. The consequences of contusion-associated bacterial lymphatic migration may vary depending on the pathogen and virulence traits selected.

The range of exoproteins and core exoproteome of 14 S. aureus isolates representing major lineages associated with asymptomatic carriage and clinical disease in the United Kingdom was identified by mass spectrometry proteomics using a combined database incorporating sequences derived from 39 S. aureus genomes. In all, 632 different proteins were identified and, of these, only 52 (8%) were found in all 14 isolates whereas 144 (23%) were found in just a single isolate. Comparison of the observed mass of each protein (based on migration by SDS-polyacrylamide electrophoresis) with its predicted mass (based on amino acid sequence) suggested that 95% of the proteins identified were not subject to any major post translational modification. Migration of 5% of proteins was not as expected: 1% of proteins migrated at a mass greater than predicted, while 4% of proteins appeared to have undergone proteolytic cleavage; these included SsaA2, Aur, SspP, Ebh as well as BlaR1, MecR1, FsH, OatA and LtaS. Intriguingly, a truncated SasG was produced by a single CC8 USA300-like strain. The analysis provided evidence of the marked heterogeneity in protein expression by S. aureus in broth, while yielding a core but narrow common exoproteome.

The complement cascade is crucial for clearance and control of invading pathogens, and as such is a key target for pathogen mediated host modulation. C3 is the central molecule of the complement cascade, and plays a vital role in opsonization of bacteria and recruitment of neutrophils to the site of infection. Streptococcal species have evolved multiple mechanisms to disrupt complement-mediated innate immunity, among which ScpA (C5a peptidase), a C5a inactivating enzyme, is widely conserved. Here we demonstrate for the first time that pyogenic streptococcal species are capable of cleaving C3, and identify C3 and C3a as novel substrates for the streptococcal ScpA, which are functionally inactivated as a result of cleavage 7 amino acids upstream of the natural C3 convertase. Cleavage of C3a by ScpA resulted in disruption of human neutrophil activation, phagocytosis and chemotaxis, while cleavage of C3 generated abnormally-sized C3a and C3b moieties with impaired function, in particular reducing C3 deposition on the bacterial surface. Despite clear effects on human complement, expression of ScpA reduced clearance of group A streptococci in vivo in wildtype and C5 deficient mice, and promoted systemic bacterial dissemination in mice that lacked both C3 and C5, suggesting an additional complement-independent role for ScpA in streptococcal pathogenesis. ScpA was shown to mediate streptococcal adhesion to both human epithelial and endothelial cells, consistent with a role in promoting bacterial invasion within the host. Taken together, these data show that ScpA is a multi-functional virulence factor with both complement-dependent and independent roles in streptococcal pathogenesis., Author summary The complement pathway is critical in the innate immune response to bacterial pathogens. It consists of a self-perpetuating proteolytic cascade initiated via three distinct pathways that converge at the central complement protein, C3. Pathogens must evade complement-mediated immunity to cause disease, and inactivation of the C3 protein can dampen all effectors of this pathway. Streptococcal species are the causative agents of an array of infections ranging from the benign to lethal. Using the human pathogen Group A Streptococcus as a representative species, we show that the enzyme ScpA, which is conserved amongst the pyogenic streptococci, cleaves human C3a and also C3, releasing abnormally sized and functionally-impaired fragments. As a result, invading streptococci were less well opsonized and host immune cells not properly activated, reducing bacterial phagocytosis and clearance. Despite manifest in vitro activity against complement factors and human neutrophils, ScpA was still able to contribute to systemic bacterial spread in mice lacking C3 and C5. ScpA was also demonstrated to mediate streptococcal adhesion to both epithelial and endothelial cells, which may enhance bacterial systemic spread. Our study highlights the likely importance of both complement-independent and complement-dependent roles for ScpA in streptococcal pathogenesis.

Background A recently-licensed 10-valent pneumococcal conjugate vaccine (PHiD-CV; Synflorix, GSK) uses Protein D from Haemophilus influenzae as a carrier protein. PHiD-CV therefore has the potential to provide additional protection against nontypeable H. influenzae (NTHi). NTHi frequently causes respiratory tract infections and is associated with significant morbidity and mortality worldwide and there is currently no vaccine. Methods We developed mouse models of NTHi infection and influenza/NTHi superinfection. Mice were immunized with PHiD-CV, heat-killed NTHi, or a 13-valent pneumococcal conjugate vaccine that did not contain Protein D (PCV13; Prevenar, Pfizer) and then infected intranasally with NTHi. Results Infection with NTHi resulted in weight loss, inflammation and airway neutrophilia. In a superinfection model, prior infection with pandemic H1N1 influenza virus (strain A/England/195/2009) augmented NTHi infection severity, even with a lower bacterial challenge dose. Immunization with PHiD-CV produced high levels of antibodies that were specific against Protein D, but not heat-killed NTHi. Immunization with PHiD-CV led to a slight reduction in bacterial load, but no change in disease outcome. Conclusions PHiD-CV induced high levels of Protein D-specific antibodies, but did not augment pulmonary clearance of NTHi. We found no evidence to suggest that PHiD-CV will offer added benefit by preventing NTHi lung infection.

Nontyphoidal strains of Salmonella are a major cause of fatal bacteremia in Africa. Developing a vaccine requires an improved understanding of the relevant mechanisms of protective immunity, and the mouse model of Salmonella infection is useful for studying immunity to Salmonella in vivo. It is important to appreciate the similarities and differences between immunity to Salmonella in mice and men. Ab is important for protection against nontyphoidal Salmonella in both species, and we have previously found an important role for Ab in cell-free complement-mediated bactericidal activity against Salmonella in Africans. It is unclear whether this modality of immunity is relevant in the mouse model. C57BL/6, BALB/c, and C3H mice immunized with heat-killed Salmonella Typhimurium strains D23580 (African invasive strain) and SL1344 and live-attenuated strain SL3261 produced a Salmonella-specific Ab response. Sera from these mice deposited reduced levels of C3 on Salmonella compared with human sera and were unable to kill both wild-type and galE− rough mutant of D23580, indicating absent cell-free killing via classical and alternative complement pathways. Supplementing immune mouse sera with human complement enabled killing of Salmonella, whereas addition of human anti-Salmonella Ab to immune mouse sera had no effect. These findings indicate that mouse serum cannot effect cell-free complement-dependent killing of Salmonella, because of the reduced mouse complement ability to kill these bacteria compared with human complement. This difference in Ab-dependent immunity to Salmonella in mice and men must be considered when applying findings from the mouse model of Salmonella disease and vaccination response to man.