Staphylococcus aureus is a major bacterial pathogen, which causes severe blood and tissue infections that frequently emerge by autoinfection with asymptomatically carried nose and skin populations. However, recent studies report that bloodstream isolates differ systematically from those found in the nose and skin, exhibiting reduced toxicity toward leukocytes. In two patients, an attenuated toxicity bloodstream infection evolved from an asymptomatically carried high-toxicity nasal strain by loss-of-function mutations in the gene encoding the transcription factor repressor of surface proteins (rsp). Here, we report that rsp knockout mutants lead to global transcriptional and proteomic reprofiling, and they exhibit the greatest signal in a genome-wide screen for genes influencing S. aureus survival in human cells. This effect is likely to be mediated in part via SSR42, a long-noncoding RNA. We show that rsp controls SSR42 expression, is induced by hydrogen peroxide, and is required for normal cytotoxicity and hemolytic activity. Rsp inactivation in laboratory- and bacteremia-derived mutants attenuates toxin production, but up-regulates other immune subversion proteins and reduces lethality during experimental infection. Crucially, inactivation of rsp preserves bacterial dissemination, because it affects neither formation of deep abscesses in mice nor survival in human blood. Thus, we have identified a spontaneously evolving, attenuated-cytotoxicity, nonhemolytic S. aureus phenotype, controlled by a pleiotropic transcriptional regulator/noncoding RNA virulence regulatory system, capable of causing S. aureus bloodstream infections. Such a phenotype could promote deep infection with limited early clinical manifestations, raising concerns that bacterial evolution within the human body may contribute to severe infection.

Community-acquired (CA) Staphylococcus aureus cause various diseases even in healthy individuals. Enhanced virulence of CA-strains is partly attributed to increased production of toxins such as phenol-soluble modulins (PSM). The pathogen is internalized efficiently by mammalian host cells and intracellular S. aureus has recently been shown to contribute to disease. Upon internalization, cytotoxic S. aureus strains can disrupt phagosomal membranes and kill host cells in a PSM-dependent manner. However, PSM are not sufficient for these processes. Here we screened for factors required for intracellular S. aureus virulence. We infected escape reporter host cells with strains from an established transposon mutant library and detected phagosomal escape rates using automated microscopy. We thereby, among other factors, identified a non-ribosomal peptide synthetase (NRPS) to be required for efficient phagosomal escape and intracellular survival of S. aureus as well as induction of host cell death. By genetic complementation as well as supplementation with the synthetic NRPS product, the cyclic dipeptide phevalin, wild-type phenotypes were restored. We further demonstrate that the NRPS is contributing to virulence in a mouse pneumonia model. Together, our data illustrate a hitherto unrecognized function of the S. aureus NRPS and its dipeptide product during S. aureus infection.

Chlamydia trachomatis, an obligate intracellular human pathogen, is a major cause of sexually transmitted diseases. Infections often occur without symptoms, a feature that has been attributed to the ability of the pathogen to evade the host immune response. We show here that C. trachomatis paralyses the host immune system by preventing the activation of polymorphic nuclear leukocytes (PMNs). PMNs infected with Chlamydia fail to produce neutrophil extracellular traps and the bacteria are able to survive in PMNs for extended periods of time. We have identified the secreted chlamydial protease-like activating factor (CPAF) as an effector mediating the evasion of the innate immune response since CPAF-deficient Chlamydia activate PMNs and are subsequently efficiently killed. CPAF suppresses the oxidative burst and interferes with chemical-mediated activation of neutrophils. We identified formyl peptide receptor 2 (FPR2) as a target of CPAF. FPR2 is cleaved by CPAF and released from the surface of PMNs. In contrast to previously described subversion mechanisms that mainly act on already activated PMNs, we describe here details of how Chlamydia actively paralyses PMNs, including the formation of neutrophil extracellular traps, to evade the host's innate immune response.

There is accumulating evidence that the lower airway microbiota impacts lung health. However, the link between microbial community composition and lung homeostasis remains elusive. We combine amplicon sequencing and bacterial culturing to characterize the viable bacterial community in 234 longitudinal bronchoalveolar lavage samples from 64 lung transplant recipients and establish links to viral loads, host gene expression, lung function, and transplant health. We find that the lung microbiota post-transplant can be categorized into four distinct compositional states, ‘pneumotypes’. The predominant ‘balanced’ pneumotype is characterized by a diverse bacterial community with moderate viral loads, and host gene expression profiles suggesting immune tolerance. The other three pneumotypes are characterized by being either microbiota-depleted, or dominated by potential pathogens, and are linked to increased immune activity, lower respiratory function, and increased risks of infection and rejection. Collectively, our findings establish a link between the lung microbial ecosystem, human lung function, and clinical stability post-transplant.

25Various bacteria of the family Acetobacteraceae are associated with the gut 26 environment of insects. Honey bees harbor two distinct Acetobacteraceae in their gut, 27Alpha2.1 and Alpha2.2. While Alpha2.1 seems to be a gut specialist, Alpha2.2 is also 28 found in the diet (e.g. royal jelly), the hypopharyngeal glands, and the larvae of honey 29 bees. Here, we combined amplicon and genome sequencing to better understand 30 functional differences associated with the ecology of Alpha2.1 and Alpha2.2. We find 31 that the two phylotypes are differentially distributed along the worker and queen bee 32 gut. Phylogenetic analysis shows that Alpha2.2 is nested within the acetic acid 33 bacteria and consists of two separate sub-lineages, whereas Alpha2.1 belongs to a 34 basal lineage with an unusual GC content for Acetobacteraceae. Gene content analysis 35 revealed major differences in the central carbon and respiratory metabolism between 36 the two phylotypes. While Alpha2.2 encodes two periplasmic dehydrogenases to 37 carry out oxidative fermentation, Alpha2.1 lacks this capability, but instead harbors 38 a diverse set of cytoplasmic dehydrogenases. These differences are accompanied by 39 the loss of the TCA cycle in Alpha2.2, but not in Alpha2.1. We speculate that Alpha2.2 40 has specialized for fast-resource utilization through incomplete carbohydrate 41 oxidation, giving it an advantage in sugar-rich environments such as royal jelly. On 42 the contrary, the broader metabolic range of Alpha2.1 may provide an advantage in 43 the worker bee hindgut, where competition with other bacteria and flexibility in 44 resource utilization may be relevant for persistence. Our results show that bacteria 45 belonging to the same family may utilize vastly different strategies to colonize niches 46 associated with the animal gut. 47 48According to a recent review on the taxonomy of Alphaproteobacteria (Munoz-49 Gomez, et al. 2019) and the standardized genome phylogeny-based taxonomy of 50 Parks et al. (Parks, et al. 2018), the family Acetobacteraceae (Rhodospirillales) is 51 comprised of an externally branching acidophilic/neutrophilic group and an internal 52 acetous group. The latter group includes acetic acid bacteria (AABs), which constitute 53 the vast majority of the described taxa of the Acetobacteraceae (Komagata, et al. 54 2014). 55 AAB inhabit sugar-rich environments and use a rather exceptional strategy to gain 56 energy. They oxidize sugars or sugar alcohols on the periplasmic side of the cell 57 envelop with the help of membrane-bound dehydrogenases that are linked to the 58 respiratory chain in a process known as oxidative fermentation (Matsushita and 59Matsutani 2016). This particular oxidative metabolism results in the accumulation of 60 fermentation products (such as acetic acid) in the environment. AABs naturally occur 61 in association with plants, flowers, and fruits (Bartowsky and Henschke 2008; 62

Stingless bees are the most diverse group of the corbiculate bees and represent important pollinator species throughout the tropics and subtropics. They harbor specialized microbial communities in their gut that are related to those found in honey bees and bumblebees and that are likely important for bee health.

Staphylococcus aureus is a common cause of bacteremia that can lead to severe complications once the bacteria exit the bloodstream and establish infection in secondary organs. Despite its clinical relevance, little is known about the bacterial factors facilitating the development of these metastatic infections. Here, we used an S. aureus transposon mutant library coupled to transposon insertion sequencing (Tn-Seq) to identify genes that are critical for efficient bacterial colonization of secondary organs in a murine model of metastatic bloodstream infection. Our transposon screen identified a LysR-type transcriptional regulator (LTTR), which was required for efficient colonization of secondary organs such as the kidneys in infected mice. The critical role of LTTR in secondary organ colonization was confirmed using an isogenic mutant deficient in the expression of LTTR. To identify the set of genes controlled by LTTR, we used an S. aureus strain carrying the LTTR gene in an inducible expression plasmid. Gene expression analysis upon induction of LTTR showed increased transcription of genes involved in branched-chain amino acid biosynthesis, a methionine sulfoxide reductase, and a copper transporter as well as decreased transcription of genes coding for urease and components of pyrimidine nucleotides. Furthermore, we show that transcription of LTTR is repressed by glucose, is induced under microaerobic conditions, and required trace amounts of copper ions. Our data thus pinpoints LTTR as an important element that enables a rapid adaptation of S. aureus to the changing host microenvironment. IMPORTANCE Staphylococcus aureus is an important pathogen that can disseminate via the bloodstream and establish metastatic infections in distant organs. To achieve a better understanding of the bacterial factors facilitating the development of these metastatic infections, we used in this study a Staphylococcus aureus transposon mutant library in a murine model of intravenous infection, where bacteria first colonize the liver as the primary infection site and subsequently progress to secondary sites such as the kidney and bones. We identified a novel LysR-type transcriptional regulator (LTTR), which was specifically required by S. aureus for efficient colonization of secondary organs. We also determined the transcriptional activation as well as the regulon of LTTR, which suggests that this regulator is involved in the metabolic adaptation of S. aureus to the host microenvironment found in secondary infection sites.

Social bees harbor conserved gut microbiota that may have been acquired in a common ancestor of social bees and subsequently co-diversified with their hosts. However, most of this knowledge is based on studies on the gut microbiota of honey bees and bumble bees. Much less is known about the gut microbiota of the third and most diverse group of social bees, the stingless bees. Specifically, the absence of genomic data from their microbiota presents an important knowledge gap in understanding the evolution and functional diversity of the social bee microbiota. Here we combined community profiling with culturing and genome sequencing of gut bacteria from six neotropical stingless bee species from Brazil. Phylogenomic analyses show that most stingless bee gut isolates form deep-branching sister clades of core members of the honey bee and bumble bee gut microbiota with conserved functional capabilities, confirming the common ancestry and ecology of their microbiota. However, our bacterial phylogenies were not congruent with those of the host indicating that the evolution of the social bee gut microbiota was not driven by strict co-diversification, but included host switches and independent symbiont gain and losses. Finally, as reported for the honey bee and bumble bee microbiota, we find substantial genomic divergence among strains of stingless bee gut bacteria suggesting adaptation to different host species and glycan niches. Our study offers first insights into the genomic diversity of the stingless bee microbiota, and highlights the need for broader samplings to understand the evolution of the social bee gut microbiota.