High Bacillary Burden and the ESX-1 Type VII Secretion System Promote MHC Class I Presentation by Mycobacterium tuberculosis-Infected Macrophages to CD8 T Cells.

We used a mouse model to study how Mycobacterium tuberculosis subverts host defenses to persist in macrophages despite immune pressure. CD4 T cells can recognize macrophages infected with a single bacillus in vitro. Under identical conditions, CD8 T cells inefficiently recognize infected macrophages and fail to restrict M. tuberculosis growth, although they can inhibit M. tuberculosis growth during high-burden intracellular infection. We show that high intracellular M. tuberculosis numbers cause macrophage death, leading other macrophages to scavenge cellular debris and cross-present the TB10.4 Ag to CD8 T cells. Presentation by infected macrophages requires M. tuberculosis to have a functional ESX-1 type VII secretion system. These data indicate that phagosomal membrane damage and cell death promote MHC class I presentation of the immunodominant Ag TB10.4 by macrophages. Although this mode of Ag presentation stimulates cytokine production that we presume would be host beneficial, killing of uninfected cells could worsen immunopathology. We suggest that shifting the focus of CD8 T cell recognition to uninfected macrophages would limit the interaction of CD8 T cells with infected macrophages and impair CD8 T cell-mediated resolution of tuberculosis.

Tetraspanin-5-mediated MHC class I clustering is required for optimal CD8 T cell activation.

MHC molecules are not randomly distributed on the plasma membrane but instead are present in discrete nanoclusters. The mechanisms that control formation of MHC I nanoclusters and the importance of such structures are incompletely understood. Here, we report a molecular association between tetraspanin-5 (Tspan5) and MHC I molecules that started in the endoplasmic reticulum and was maintained on the plasma membrane. This association was observed both in mouse dendritic cells and in human cancer cell lines. Loss of Tspan5 reduced the size of MHC I clusters without affecting MHC I peptide loading, delivery of complexes to the plasma membrane, or overall surface MHC I levels. Functionally, CD8 T cell responses to antigen presented by Tspan5-deficient dendritic cells were impaired but were restored by antibody-induced reclustering of MHC I molecules. In contrast, Tspan5 did not associate with two other plasma membrane proteins, Flotillin1 and CD55, with or the endoplasmic reticulum proteins Tapasin and TAP. Thus, our findings identify a mechanism underlying the clustering of MHC I molecules that is important for optimal T cell responses.

Mycobacterium tuberculosis Evasion of Guanylate Binding Protein-Mediated Host Defense in Mice Requires the ESX1 Secretion System.

Cell-intrinsic immune mechanisms control intracellular pathogens that infect eukaryotes. The intracellular pathogen Mycobacterium tuberculosis (Mtb) evolved to withstand cell-autonomous immunity to cause persistent infections and disease. A potent inducer of cell-autonomous immunity is the lymphocyte-derived cytokine IFNγ. While the production of IFNγ by T cells is essential to protect against Mtb, it is not capable of fully eradicating Mtb infection. This suggests that Mtb evades a subset of IFNγ-mediated antimicrobial responses, yet what mechanisms Mtb resists remains unclear. The IFNγ-inducible Guanylate binding proteins (GBPs) are key host defense proteins able to control infections with intracellular pathogens. GBPs were previously shown to directly restrict Mycobacterium bovis BCG yet their role during Mtb infection has remained unknown. Here, we examine the importance of a cluster of five GBPs on mouse chromosome 3 in controlling Mycobacterial infection. While M. bovis BCG is directly restricted by GBPs, we find that the GBPs on chromosome 3 do not contribute to the control of Mtb replication or the associated host response to infection. The differential effects of GBPs during Mtb versus M. bovis BCG infection is at least partially explained by the absence of the ESX1 secretion system from M. bovis BCG, since Mtb mutants lacking the ESX1 secretion system become similarly susceptible to GBP-mediated immune defense. Therefore, this specific genetic interaction between the murine host and Mycobacteria reveals a novel function for the ESX1 virulence system in the evasion of GBP-mediated immunity.

DNA Barcoding Reveals Generalization and Host Overlap in Hummingbird Flower Mites: Implications for the Mating Rendezvous Hypothesis.

AbstractHummingbird flower mites are assumed to monopolize single host plant species owing to sexual selection for unique mating rendezvous sites. We tested the main assumption of the mating rendezvous hypothesis-extreme host specialization-by reconstructing interactions among tropical hummingbird flower mites and their host plants using DNA barcoding and taxonomic identifications. We collected 10,654 mites from 489 flowers. We extracted DNA from 1,928 mite specimens and amplified the cytochrome c oxidase I (CO1) DNA barcode. We analyzed the network structure to assess the degree of generalization or specialization of mites to their host plants. We recorded 18 species of hummingbird flower mites from three genera (Proctolaelaps, Rhinoseius, and Tropicoseius) interacting with 14 species of plants. We found that generalist mites are common, and congeneric mite species often share host plants. Our results challenge the assumption of strict specialization that supports this system as an example of mating rendezvous evolution.

CD11cHi monocyte-derived macrophages are a major cellular compartment infected by Mycobacterium tuberculosis.

During tuberculosis, lung myeloid cells have two opposing roles: they are an intracellular niche occupied by Mycobacterium tuberculosis, and they restrict bacterial replication. Lung myeloid cells from mice infected with yellow-fluorescent protein expressing M. tuberculosis were analyzed by flow cytometry and transcriptional profiling to identify the cell types infected and their response to infection. CD14, CD38, and Abca1 were expressed more highly by infected alveolar macrophages and CD11cHi monocyte-derived cells compared to uninfected cells. CD14, CD38, and Abca1 "triple positive" (TP) cells had not only the highest infection rates and bacterial loads, but also a strong interferon-γ signature and nitric oxide synthetase-2 production indicating recognition by T cells. Despite evidence of T cell recognition and appropriate activation, these TP macrophages are a cellular compartment occupied by M. tuberculosis long-term. Defining the niche where M. tuberculosis resists elimination promises to provide insight into why inducing sterilizing immunity is a formidable challenge.

Deconvolution microscopy: A platform for rapid on-site evaluation of fine needle aspiration specimens that enables recovery of the sample.

CONTEXT: Rapid on-site evaluation (ROSE) optimises the performance of cytology, but requires skilled handling, and smearing can make the material unavailable for some ancillary tests. There is a need to facilitate ROSE without sacrificing part of the sample. OBJECTIVE: We evaluated the image quality of inexpensive deconvolution fluorescence microscopy for optically sectioning non-smeared fine needle aspiration (FNA) tissue fragments. DESIGN: A portion of residual material from 14 FNA samples was stained for 3 min in Hoechst 33342 and Sypro™ Red to label DNA and protein respectively, transferred to an imaging chamber, and imaged at 200× or 400× magnification at 1 micron intervals using a GE DeltaVision inverted fluorescence microscope. A deconvolution algorithm was applied to remove out-of-plane signal, and the resulting images were inverted and pseudocoloured to resemble H&E sections. Five cytopathologists blindly diagnosed 2 to 4 representative image stacks per case (total 70 evaluations), and later compared them to conventional epifluorescent images. RESULTS: Accurate definitive diagnoses were rendered in 45 (64%) of 70 total evaluations; equivocal diagnoses (atypical or suspicious) were made in 21 (30%) of the 70. There were two false positive and two false negative "definite" diagnoses in three cases (4/70; 6%). Cytopathologists preferred deconvolved images compared to raw images (P < 0.01). The imaged fragments were recovered and prepared into a ThinPrep or cell block without discernible alteration. CONCLUSIONS: Deconvolution improves image quality of FNA fragments compared to epifluorescence, often allowing definitive diagnosis while enabling the ROSE material to be subsequently triaged.

A Developmental Analysis of Juxtavascular Microglia Dynamics and Interactions with the Vasculature.

Microglia, a resident CNS macrophage, are dynamic cells, constantly extending and retracting their processes as they contact and functionally regulate neurons and other glial cells. There is far less known about microglia-vascular interactions, particularly under healthy steady-state conditions. Here, we use the male and female mouse cerebral cortex to show that a higher percentage of microglia associate with the vasculature during the first week of postnatal development compared with older ages and that the timing of these associations is dependent on the fractalkine receptor (CX3CR1). Similar developmental microglia-vascular associations were detected in the human brain. Using live imaging in mice, we found that juxtavascular microglia migrated when microglia are actively colonizing the cortex and became stationary by adulthood to occupy the same vascular space for nearly 2 months. Further, juxtavascular microglia at all ages associate with vascular areas void of astrocyte endfeet, and the developmental shift in microglial migratory behavior along vessels corresponded to when astrocyte endfeet more fully ensheath vessels. Together, our data provide a comprehensive assessment of microglia-vascular interactions. They support a mechanism by which microglia use the vasculature to migrate within the developing brain parenchyma. This migration becomes restricted on the arrival of astrocyte endfeet such that juxtavascular microglia become highly stationary and stable in the mature cortex.SIGNIFICANCE STATEMENT We report the first extensive analysis of juxtavascular microglia in the healthy, developing, and adult brain. Live imaging revealed that juxtavascular microglia within the cortex are highly motile and migrate along vessels as they are colonizing cortical regions. Using confocal, expansion, super-resolution, and electron microscopy, we determined that microglia associate with the vasculature at all ages in areas lacking full astrocyte endfoot coverage and motility of juxtavascular microglia ceases as astrocyte endfeet more fully ensheath the vasculature. Our data lay the fundamental groundwork to investigate microglia-astrocyte cross talk and juxtavascular microglial function in the healthy and diseased brain. They further provide a potential mechanism by which vascular interactions facilitate microglial colonization of the brain to later regulate neural circuit development.

Oscillating calcium signals in smooth muscle cells underlie the persistent basal tone of internal anal sphincter.

A persistent basal tone in the internal anal sphincter (IAS) is essential for keeping the anal canal closed and fecal continence; its inhibition via the rectoanal inhibitory reflex (RAIR) is required for successful defecation. However, cellular signals underlying the IAS basal tone remain enigmatic. Here we report the origin and molecular mechanisms of calcium signals that control the IAS basal tone, using a combination approach including a novel IAS slice preparation that retains cell arrangement and architecture as in vivo, 2-photon imaging, and cell-specific gene-modified mice. We found that IAS smooth muscle cells generate two forms of contractions (i.e., phasic and sustained contraction) and Ca2+ signals (i.e., synchronized Ca2+ oscillations [SCaOs] and asynchronized Ca2+ oscillations [ACaOs]) that last for hours. RyRs, TMEM16A, L-type Ca2+ channels, and gap junctions are required for SCaOs, which account for phasic contraction and 75% of sustained contraction. Nevertheless, only RyRs are required for ACaOs, which contribute 25% of sustained contraction. Nitric oxide, the primary neurotransmitter mediating the RAIR, blocks both types of Ca2+ signals, leading to IAS's full relaxation. Our results show that the oscillating nature of Ca2+ signals generates and maintains the basal tone without causing cytotoxicity to IAS. Our study provides insight into fecal continence and normal defecation.

Demographic Attritions, Elevational Refugia, and the Resilience of Insect Populations to Projected Global Warming.

AbstractTropical mountains might protect species from global warming by facilitating biotic migrations upslope. Current predictions of tropical biotic responses to global warming are based on correlations between species elevational distributions and temperatures. Because biotic attritions, range shifts, and mountaintop extinctions result from complex demographic processes, predictive models must be based on mechanistic associations between temperature and fitness. Our study combines long-term temperature records with experimental demography to determine the contribution of local adaptation to organismal resilience in a warming world. On the Barva volcano in Costa Rica, Cephaloleia belti (Coleoptera: Chrysomelidae) displays high-elevation (960-2,100 m asl) and low-elevation (50-960 m asl) mitochondrial haplotypes. We reared haplotype cohorts at temperatures prevalent along the elevational gradient (i.e., 10°-30°C). Based on ambient temperatures recorded every half hour for 4 years, we projected average instantaneous population growth rates ([Formula: see text]) at current and future temperatures (i.e., +1° to 6°C) for each beetle haplotype. Haplotypes are adapted to local temperatures, but with a temperature increase beyond 2°C, both haplotypes will face lower-elevation demographic attritions and extinctions. Upper distribution limits serve as potential elevational refugia from global warming. This study shows how species resilience to global warming emerges from complex fitness responses of locally adapted phenotypes facing novel environments.

Real-time imaging of integrin ß4 dynamics using a reporter cell line generated by Crispr/Cas9 genome editing.

The ability to monitor changes in the expression and localization of integrins is essential for understanding their contribution to development, tissue homeostasis and disease. Here, we pioneered the use of Crispr/Cas9 genome editing to tag an allele of the ß4 subunit of the α6ß4 integrin. A tdTomato tag was inserted with a linker at the C-terminus of integrin ß4 in mouse mammary epithelial cells. Cells harboring this tagged allele were similar to wild-type cells with respect to integrin ß4 surface expression, association with the α6 subunit, adhesion to laminin and consequent signaling. These integrin ß4 reporter cells were transformed with YAP (also known as YAP1), which enabled us to obtain novel insight into integrin ß4 dynamics in response to a migratory stimulus (scratch wound) by live-cell video microscopy. An increase in integrin ß4 expression in cells proximal to the wound edge was evident, and a population of integrin ß4-expressing cells that exhibited unusually rapid migration was identified. These findings could shed insight into integrin ß4 dynamics during invasion and metastasis. Moreover, these integrin ß4 reporter cells should facilitate studies on the contribution of this integrin to mammary gland biology and cancer.This article has an associated First Person interview with the first author of the paper.

Positive genetic covariance and limited thermal tolerance constrain tropical insect responses to global warming.

Tropical ectotherms are particularly vulnerable to global warming because their physiologies are assumed to be adapted to narrow temperature ranges. This study explores three mechanisms potentially constraining thermal adaptation to global warming in tropical insects: (a) Trade-offs in genotypic performance at different temperatures (the jack-of-all-trades hypothesis), (b) positive genetic covariance in performance, with some genotypes performing better than others at viable temperatures (the 'winner' and 'loser' genotypes hypothesis), or (c) limited genetic variation as the potential result of relaxed selection and the loss of genes associated with responses to extreme temperatures (the gene decay hypothesis). We estimated changes in growth and survival rates at multiple temperatures for three tropical rain forest insect herbivores (Cephaloleia rolled-leaf beetles, Chrysomelidae). We reared 2,746 individuals in a full sibling experimental design, at temperatures known to be experienced by this genus of beetles in nature (i.e. 10-35°C). Significant genetic covariance was positive for 16 traits, supporting the 'winner' and 'loser' genotypes hypothesis. Only two traits displayed negative cross-temperature performance correlations. We detected a substantial contribution of genetic variance in traits associated with size and mass (0%-44%), but low heritability in plastic traits such as development time (0%-6%) or survival (0%-4%). Lowland insect populations will most likely decline if current temperatures increase between 2 and 5°C. It is concerning that local adaption is already lagging behind current temperatures. The consequences of maintaining the current global warming trajectory would be devastating for tropical insects. However, if humans can limit or slow warming, many tropical ectotherms might persist in their current locations and potentially adapt to warmer temperatures.

Spatially distinct and metabolically active membrane domain in mycobacteria.

Protected from host immune attack and antibiotic penetration by their unique cell envelope, mycobacterial pathogens cause devastating human diseases such as tuberculosis. Seamless coordination of cell growth with cell envelope elongation at the pole maintains this barrier. Unraveling this spatiotemporal regulation is a potential strategy for controlling mycobacterial infections. Our biochemical analysis previously revealed two functionally distinct membrane fractions in Mycobacterium smegmatis cell lysates: plasma membrane tightly associated with the cell wall (PM-CW) and a distinct fraction of pure membrane free of cell wall components (PMf). To provide further insight into the functions of these membrane fractions, we took the approach of comparative proteomics and identified more than 300 proteins specifically associated with the PMf, including essential enzymes involved in cell envelope synthesis such as a mannosyltransferase, Ppm1, and a galactosyltransferase, GlfT2. Furthermore, comparative lipidomics revealed the distinct lipid composition of the PMf, with specific association of key cell envelope biosynthetic precursors. Live-imaging fluorescence microscopy visualized the PMf as patches of membrane spatially distinct from the PM-CW and notably enriched in the pole of the growing cells. Taken together, our study provides the basis for assigning the PMf as a spatiotemporally distinct and metabolically active membrane domain involved in cell envelope biogenesis.

New insights into TB physiology suggest untapped therapeutic opportunities.

The current regimens used to treat tuberculosis are largely comprised of serendipitously discovered drugs that are combined based on clinical experience. Despite curing millions, these drug regimens are limited by the long course of therapy, the emergence of resistance, and the persistent tissue damage that remains after treatment. The last two decades have produced only a single new drug but have represented a renaissance in our understanding of the physiology of tuberculosis infection. The advent of mycobacterial genetics, sophisticated immunological methods, and imaging technologies have transformed our understanding of bacterial physiology as well as the contribution of the host response to disease outcome. Specific alterations in bacterial metabolism, heterogeneity in bacterial state, and drug penetration all limit the effectiveness of antimicrobial therapy. This review summarizes these new biological insights and discusses strategies to exploit them for the rational development of more effective therapeutics. Three general strategies are discussed. First, our emerging insight into bacterial physiology suggests new pathways that might be targeted to accelerate therapy. Second, we explore whether the concept of genetic synergy can be used to design effective combination therapies. Finally, we outline possible approaches to modulate the host response to accentuate antibiotic efficacy. These biology-driven strategies promise to produce more effective therapies.

Structural and Genetic Analyses of the Mycobacterium tuberculosis Protein Kinase B Sensor Domain Identify a Potential Ligand-binding Site.

Monitoring the environment with serine/threonine protein kinases is critical for growth and survival of Mycobacterium tuberculosis, a devastating human pathogen. Protein kinase B (PknB) is a transmembrane serine/threonine protein kinase that acts as an essential regulator of mycobacterial growth and division. The PknB extracellular domain (ECD) consists of four repeats homologous to penicillin-binding protein and serine/threonine kinase associated (PASTA) domains, and binds fragments of peptidoglycan. These properties suggest that PknB activity is modulated by ECD binding to peptidoglycan substructures, however, the molecular mechanisms underpinning PknB regulation remain unclear. In this study, we report structural and genetic characterization of the PknB ECD. We determined the crystal structures of overlapping ECD fragments at near atomic resolution, built a model of the full ECD, and discovered a region on the C-terminal PASTA domain that has the properties of a ligand-binding site. Hydrophobic interaction between this surface and a bound molecule of citrate was observed in a crystal structure. Our genetic analyses in M. tuberculosis showed that nonfunctional alleles were produced either by deletion of any of single PASTA domain or by mutation of individual conserved residues lining the putative ligand-binding surface of the C-terminal PASTA repeat. These results define two distinct structural features necessary for PknB signal transduction, a fully extended ECD and a conserved, membrane-distal putative ligand-binding site.

Phosphorylation of the Peptidoglycan Synthase PonA1 Governs the Rate of Polar Elongation in Mycobacteria.

Cell growth and division are required for the progression of bacterial infections. Most rod-shaped bacteria grow by inserting new cell wall along their mid-section. However, mycobacteria, including the human pathogen Mycobacterium tuberculosis, produce new cell wall material at their poles. How mycobacteria control this different mode of growth is incompletely understood. Here we find that PonA1, a penicillin binding protein (PBP) capable of transglycosylation and transpeptidation of cell wall peptidoglycan (PG), is a major governor of polar growth in mycobacteria. PonA1 is required for growth of Mycobacterium smegmatis and is critical for M. tuberculosis during infection. In both cases, PonA1's catalytic activities are both required for normal cell length, though loss of transglycosylase activity has a more pronounced effect than transpeptidation. Mutations that alter the amount or the activity of PonA1 result in abnormal formation of cell poles and changes in cell length. Moreover, altered PonA1 activity results in dramatic differences in antibiotic susceptibility, suggesting that a balance between the two enzymatic activities of PonA1 is critical for survival. We also find that phosphorylation of a cytoplasmic region of PonA1 is required for normal activity. Mutations in a critical phosphorylated residue affect transglycosylase activity and result in abnormal rates of cell elongation. Together, our data indicate that PonA1 is a central determinant of polar growth in mycobacteria, and its governance of cell elongation is required for robust cell fitness during both host-induced and antibiotic stress.

Subpolar addition of new cell wall is directed by DivIVA in mycobacteria.

Mycobacteria are surrounded by a complex multilayered envelope and elongate at the poles. The principles that organize the coordinated addition of chemically diverse cell wall layers during polar extension remain unclear. We show that enzymes mediating the terminal cytosolic steps of peptidoglycan, arabinogalactan, and mycolic acid synthesis colocalize at sites of cell growth or division. The tropomyosin-like protein, DivIVA, is targeted to the negative curvature of the pole, is enriched at the growing end, and determines cell shape from this site. In contrast, cell wall synthetic complexes are concentrated at a distinct subpolar location. When viewed at subdiffraction resolution, new peptidoglycan is deposited at this subpolar site, and inert cell wall covers the DivIVA-marked tip. The differentiation between polar tip and cell wall synthetic complexes is also apparent at the biochemical level. Enzymes that generate mycolate precursors interact with DivIVA, but the final condensation of mycolic acids occurs in a distinct protein complex at the site of nascent cell wall addition. We propose an ultrastructural model of mycobacterial polar growth where new cell wall is added in an annular zone below the cell tip. This model may be broadly applicable to other bacterial and fungal organisms that grow via polar extension.

Ode to Ehrlich and Raven or how herbivorous insects might drive plant speciation.

Fifty years ago, Ehrlich and Raven proposed that insect herbivores have driven much of plant speciation, particularly at tropical latitudes. There have been no explicit tests of their hypotheses. Indeed there were no proposed mechanisms either at the time or since by which herbivores might generate new plant species. Here we outline two main classes of mechanisms, prezygotic and postzygotic, with a number of scenarios in each by which herbivore-driven changes in host plant secondary chemistry might lead to new plant lineage production. The former apply mainly to a sympatric model of speciation while the latter apply to a parapatric or allopatric model. Our review suggests that the steps of each mechanism are known to occur individually in many different systems, but no scenario has been thoroughly investigated in any one system. Nevertheless, studies of Dalechampia and its herbivores and pollinators, and patterns of defense tradeoffs in trees on different soil types in the Peruvian Amazon provide evidence consistent with the original hypotheses of Ehrlich and Raven. For herbivores to drive sympatric speciation, our findings suggest that interactions with both their herbivores and their pollinators should be considered. In contrast, herbivores may drive speciation allopatrically without any influence by pollinators. Finally, there is evidence that these mechanisms are more likely to occur at low latitudes and thus more likely to produce new species in the tropics. The mechanisms we outline provide a predictive framework for further study of the general role that herbivores play in diversification of their host plants.

Osmosensory signaling in Mycobacterium tuberculosis mediated by a eukaryotic-like Ser/Thr protein kinase.

Bacteria are able to adapt to dramatically different microenvironments, but in many organisms, the signaling pathways, transcriptional programs, and downstream physiological changes involved in adaptation are not well-understood. Here, we discovered that osmotic stress stimulates a signaling network in Mycobacterium tuberculosis regulated by the eukaryotic-like receptor Ser/Thr protein kinase PknD. Expression of the PknD substrate Rv0516c was highly induced by osmotic stress. Furthermore, Rv0516c disruption modified peptidoglycan thickness, enhanced antibiotic resistance, and activated genes in the regulon of the alternative σ-factor SigF. Phosphorylation of Rv0516c regulated the abundance of EspA, a virulence-associated substrate of the type VII ESX-1 secretion system. These findings identify an osmosensory pathway orchestrated by PknD, Rv0516c, and SigF that enables adaptation to osmotic stress through cell wall remodeling and virulence factor production. Given the widespread occurrence of eukaryotic-like Ser/Thr protein kinases in bacteria, these proteins may play a broad role in bacterial osmosensing.

Biochemical and spatial coincidence in the provisional Ser/Thr protein kinase interaction network of Mycobacterium tuberculosis.

Many Gram-positive bacteria coordinate cellular processes by signaling through Ser/Thr protein kinases (STPKs), but the architecture of these phosphosignaling cascades is unknown. To investigate the network structure of a prokaryotic STPK system, we comprehensively explored the pattern of signal transduction in the Mycobacterium tuberculosis Ser/Thr kinome. Autophosphorylation is the dominant mode of STPK activation, but the 11 M. tuberculosis STPKs also show a specific pattern of efficient cross-phosphorylation in vitro. The biochemical specificity intrinsic to each kinase domain was used to map the provisional signaling network, revealing a three-layer architecture that includes master regulators, signal transducers, and terminal substrates. Fluorescence microscopy revealed that the STPKs are specifically localized in the cell. Master STPKs are concentrated at the same subcellular sites as their substrates, providing additional support for the biochemically defined network. Together, these studies imply a branched functional architecture of the M. tuberculosis Ser/Thr kinome that could enable horizontal signal spreading. This systems-level approach provides a biochemical and spatial framework for understanding Ser/Thr phospho-signaling in M. tuberculosis, which differs fundamentally from previously defined linear histidine kinase cascades.