2D and 3D cryo-electron microscopy, together with adsorption kinetics assays of ϕCb13 and ϕCbK phage-infected Caulobacter crescentus , provides insight into the mechanisms of infection. ϕCb13 and ϕCbK actively interact with the flagellum and subsequently attach to receptors on the cell pole. We present evidence that the first interaction of the phage with the bacterial flagellum takes place through a filament on the phage head. This contact with the flagellum facilitates concentration of phage particles around the receptor (i.e., the pilus portals) on the bacterial cell surface, thereby increasing the likelihood of infection. Phage head filaments have not been well characterized and their function is described here. Phage head filaments may systematically underlie the initial interactions of phages with their hosts in other systems and possibly represent a widespread mechanism of efficient phage propagation.

Streptococcus pneumoniae serotype 3 remains a significant cause of morbidity and mortality worldwide, despite inclusion in the 13-valent pneumococcal conjugate vaccine (PCV13). Serotype 3 increased in carriage since the implementation of PCV13 in the USA, while invasive disease rates remain unchanged. We investigated the persistence of serotype 3 in carriage and disease, through genomic analyses of a global sample of 301 serotype 3 isolates of the Netherlands3–31 (PMEN31) clone CC180, combined with associated patient data and PCV utilization among countries of isolate collection. We assessed phenotypic variation between dominant clades in capsule charge (zeta potential), capsular polysaccharide shedding, and susceptibility to opsonophagocytic killing, which have previously been associated with carriage duration, invasiveness, and vaccine escape. We identified a recent shift in the CC180 population attributed to a lineage termed Clade II, which was estimated by Bayesian coalescent analysis to have first appeared in 1968 [95% HPD: 1939–1989] and increased in prevalence and effective population size thereafter. Clade II isolates are divergent from the pre-PCV13 serotype 3 population in non-capsular antigenic composition, competence, and antibiotic susceptibility, the last of which resulting from the acquisition of a Tn916-like conjugative transposon. Differences in recombination rates among clades correlated with variations in the ATP-binding subunit of Clp protease, as well as amino acid substitutions in the comCDE operon. Opsonophagocytic killing assays elucidated the low observed efficacy of PCV13 against serotype 3. Variation in PCV13 use among sampled countries was not independently correlated with the CC180 population shift; therefore, genotypic and phenotypic differences in protein antigens and, in particular, antibiotic resistance may have contributed to the increase of Clade II. Our analysis emphasizes the need for routine, representative sampling of isolates from disperse geographic regions, including historically under-sampled areas. We also highlight the value of genomics in resolving antigenic and epidemiological variations within a serotype, which may have implications for future vaccine development.

Tuberculosis (TB) remains a serious global public health problem that results in up to 2 million deaths each year. TB is caused by the human pathogen, Mycobacterium tuberculosis (Mtb), which infects primarily innate immune cells patrolling the lung. Innate immune cells serve as barometers of the immune response against Mtb infection by determining the inflammatory milieu in the lungs and promoting the generation of adaptive immune responses. However, innate immune cells are also potential niches for bacterial replication and are readily manipulated by Mtb. Our understanding of the early interactions between Mtb and innate immune cells is limited, especially in the context of human infection. This review will focus on Mtb interactions with human macrophages, dendritic cells, neutrophils, and NK cells and detail evidence that Mtb modulation of these cells negatively impacts Mtb-specific immune responses. Furthermore, this review will emphasize important innate immune pathways uncovered through human immunogenetic studies. Insights into the human innate immune response to Mtb infection are necessary for providing a rational basis for the augmentation of immune responses against Mtb infection, especially with respect to the generation of effective anti-TB immunotherapeutics and vaccines.

e Nitric oxide (NO) is a diffusible radical gas produced from the activity of nitric oxide synthase (NOS). NOS activity in murine macrophages has a protective role against mycobacteria through generation of reactive nitrogen intermediates (RNIs). However, the production of NO by human macrophages has remained unclear due to the lack of sensitive reagents to detect NO directly. The purpose of this study was to investigate NO production and the consequence to mycobacteria in primary human macrophages. We found that Mycobacterium bovis BCG or Mycobacterium tuberculosis infection of human macrophages induced expression of NOS2 and NOS3 that resulted in detectable production of NO. Treatment with gamma interferon (IFN-␥), L-arginine, and tetrahydrobiopterin enhanced expression of NOS2 and NOS3 isoforms, as well as NO production. Both of these enzymes were shown to contribute to NO production. The maximal level of NO produced by human macrophages was not bactericidal or bacteriostatic to M. tuberculosis or BCG. The number of viable mycobacteria was increased in macrophages that produced NO, and this requires expression of nitrate reductase. An narG mutant of M. tuberculosis persisted but was unable to grow in human macrophages. Taken together, these data (i) enhance our understanding of primary human macrophage potential to produce NO, (ii) demonstrate that the level of RNIs produced in response to IFN-␥ in vitro is not sufficient to limit intracellular mycobacterial growth, and (iii) suggest that mycobacteria may use RNIs to enhance their survival in human macrophages.

Mycobacterium tuberculosis (Mtb) employs multiple strategies to evade host immune responses and persist within macrophages. We have previously shown that the cell envelope-associated Mtb serine hydrolase, Hip1, prevents robust macrophage activation and dampens host pro-inflammatory responses, allowing Mtb to delay immune detection and accelerate disease progression. We now provide key mechanistic insights into the molecular and biochemical basis of Hip1 function. We establish that Hip1 is a serine protease with activity against protein and peptide substrates. Further, we show that the Mtb GroEL2 protein is a direct substrate of Hip1 protease activity. Cleavage of GroEL2 is specifically inhibited by serine protease inhibitors. We mapped the cleavage site within the N-terminus of GroEL2 and confirmed that this site is required for proteolysis of GroEL2 during Mtb growth. Interestingly, we discovered that Hip1-mediated cleavage of GroEL2 converts the protein from a multimeric to a monomeric form. Moreover, ectopic expression of cleaved GroEL2 monomers into the hip1 mutant complemented the hyperinflammatory phenotype of the hip1 mutant and restored wild type levels of cytokine responses in infected macrophages. Our studies point to Hip1-dependent proteolysis as a novel regulatory mechanism that helps Mtb respond rapidly to changing host immune environments during infection. These findings position Hip1 as an attractive target for inhibition for developing immunomodulatory therapeutics against Mtb.

It is well accepted that pathogens can evade recognition and elimination by the host immune system by varying their antigenic targets. Thus, it has become a truism that host immunity is a major driver and determinant of the antigenic diversity of pathogens. However, it remains puzzling how host immunity selects for antigenic diversity at the level of the population, given that hosts have acquired immune responses to multiple antigens of most pathogens — sometimes through multiple effectors of both humoral and cellular immunity. In this Opinion article, we address this puzzle and the related question of why pathogens often have diversity at multiple antigenic loci. Here, we propose five hypotheses to explain the polymorphism of multiple antigens in a single species and highlight research relevant to our current models of thinking about multi-locus antigenic diversity.

44Background: Several Streptococcus pneumoniae proteins play a role in pathogenesis

successfully subverts the host immune response to promote disease progression. In addition to its known intracellular niche in macrophages, interferes with the functions of dendritic cells (DCs), which are the primary antigen-presenting cells of the immune system. We previously showed that dampens proinflammatory responses and impairs DC functions through the cell envelope-associated serine protease Hip1. Here we present data showing that GroEL2, a substrate of Hip1, modulates DC functions. The full-length GroEL2 protein elicited robust proinflammatory responses from DCs and promoted DC maturation and antigen presentation to T cells. In contrast, the cleaved form of GroEL2, which predominates in, was poorly immunostimulatory and was unable to promote DC maturation and antigen presentation. Moreover, DCs exposed to full-length, but not cleaved, GroEL2 induced strong antigen-specific gamma interferon (IFN-γ), interleukin-2 (IL-2), and IL-17A cytokine responses from CD4 T cells. Moreover, the expression of cleaved GroEL2 in the mutant restored the robust T cell responses to wild-type levels, suggesting that proteolytic cleavage of GroEL2 allows to prevent optimal DC-T cell cross talk during infection.