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Variability in bacterial sterilization is a key feature of Mycobacterium tuberculosis (Mtb) disease. In a population of human macrophages, there are macrophages that restrict Mtb growth and those that do not. However, the sources of heterogeneity in macrophage state during Mtb infection are poorly understood. Here, we perform RNAseq on restrictive and permissive macrophages and reveal that the expression of genes involved in GM-CSF signaling discriminates between the two subpopulations. We demonstrate that blocking GM-CSF makes macrophages more permissive of Mtb growth while addition of GM-CSF increases bacterial control. In parallel, we find that the loss of bacterial control that occurs in HIV-Mtb coinfected macrophages correlates with reduced GM-CSF secretion. Treatment of coinfected cells with GM-CSF restores bacterial control. Thus, we leverage the natural variation in macrophage control of Mtb to identify a critical cytokine response for regulating Mtb survival and identify components of the antimicrobial response induced by GM-CSF.

While a third of the world carries the burden of tuberculosis, disease control has been hindered by a lack of tools, including a rapid, point-of-care diagnostic and a protective vaccine. In many infectious diseases, antibodies (Abs) are powerful biomarkers and important immune mediators. However, in Mycobacterium tuberculosis (Mtb) infection, a discriminatory or protective role for humoral immunity remains unclear. Using an unbiased antibody profiling approach, we show that individuals with latent tuberculosis infection (Ltb) and active tuberculosis disease (Atb) have distinct Mtb-specific humoral responses, such that Ltb infection is associated with unique Ab Fc functional profiles, selective binding to FcγRIII, and distinct Ab glycosylation patterns. Moreover, compared to Abs from Atb, Abs from Ltb drove enhanced phagolysosomal maturation, inflammasome activation, and, most importantly, macrophage killing of intracellular Mtb. Combined, these data point to a potential role for Fc-mediated Ab effector functions, tuned via differential glycosylation, in Mtb control., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The activation of an immune response in tomato (Solanum lycopersicum) against Pseudomonas syringae relies on the recognition of E3 ligase-deficient forms of AvrPtoB by the host protein kinase, Fen. To investigate the mechanisms by which Fen-mediated immunity is regulated, we characterize in this study a Fen-interacting protein, Fni3, and its cofactor, S. lycoperiscum Uev (Suv). Fni3 encodes a homolog of the Ubc13-type ubiquitin-conjugating enzyme that catalyzes exclusively Lys-63-linked ubiquitination, whereas Suv is a ubiquitin-conjugating enzyme variant. The C-terminal region of Fen was necessary for interaction with Fni3, and this interaction was required for cell death triggered by overexpression of Fen in Nicotiana benthamiana leaves. Fni3 was shown to be an active E2 enzyme, but Suv displayed no ubiquitin-conjugating activity; Fni3 and Suv together directed Lys-63-linked ubiquitination. Decreased expression of Fni3, another tomato Ubc13 homolog, Sl-Ubc13-2, or Suv in N. benthamiana leaves diminished cell death associated with Fen-mediated immunity and cell death elicited by several other resistance (R) proteins and their cognate effectors. We also discovered that coexpression of Fen and other R proteins/effectors with a Fni3 mutant that is compromised for ubiquitin-conjugating activity diminished the cell death. These results suggest that Fni3/Sl-Ubc13-2 and Suv regulate the immune response mediated by Fen and other R proteins through Lys-63-linked ubiquitination.

Mycobacterium tuberculosis persists within macrophages in an arrested phagosome and depends upon necrosis to elude immunity and disseminate. Although apoptosis of M. tuberculosis-infected macrophages is associated with reduced bacterial growth, the bacteria are relatively resistant to other forms of death, leaving the mechanism underlying this observation unresolved. We find that after apoptosis, M. tuberculosis-infected macrophages are rapidly taken up by uninfected macrophages through efferocytosis, a dedicated apoptotic cell engulfment process. Efferocytosis of M. tuberculosis sequestered within an apoptotic macrophage further compartmentalizes the bacterium and delivers it along with the apoptotic cell debris to the lysosomal compartment. M. tuberculosis is killed only after efferocytosis, indicating that apoptosis itself is not intrinsically bactericidal but requires subsequent phagocytic uptake and lysosomal fusion of the apoptotic body harboring the bacterium. While efferocytosis is recognized as a constitutive housekeeping function of macrophages, these data indicate that it can also function as an antimicrobial effector mechanism., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Many bacterial pathogens of plants and animals use a type III secretion system to deliver diverse virulence-associated 'effector' proteins into the host cell. The mechanisms by which these effectors act are mostly unknown; however, they often promote disease by suppressing host immunity. One type III effector, AvrPtoB, expressed by the plant pathogen Pseudomonas syringae pv. tomato, has a carboxy-terminal domain that is an E3 ubiquitin ligase. Deletion of this domain allows an amino-terminal region of AvrPtoB (AvrPtoB(1-387)) to be detected by certain tomato varieties leading to immunity-associated programmed cell death. Here we show that a host kinase, Fen, physically interacts with AvrPtoB(1-387 )and is responsible for activating the plant immune response. The AvrPtoB E3 ligase specifically ubiquitinates Fen and promotes its degradation in a proteasome-dependent manner. This degradation leads to disease susceptibility in Fen-expressing tomato lines. Various wild species of tomato were found to exhibit immunity in response to AvrPtoB(1-387 )and not to full-length AvrPtoB. Thus, by acquiring an E3 ligase domain, AvrPtoB has thwarted a highly conserved host resistance mechanism.

In the summers of 2000 and 2001, tomato plants (Lycopersicon esculentum) with symptoms of stunting, curling, and marginal chlorosis of leaves, reduced leaf size, and marked reduction in fruit number, similar to those caused by Tomato yellow leaf curl virus (TYLCV), were seen in Henderson County, NC. In 2001, symptomatic plants appeared in a 40-A (18.2 ha) field in 12 foci of ≈12 plants each, at a total incidence of less than 1%. In August 2001, DNA was extracted from leaf samples from four symptomatic plants and tested by polymerase chain reaction (PCR) amplification for the presence of one or more geminiviruses. Two sets of primers were used to test for begomoviruses, AC1048 and PCRv181 (3,4), which amplify a 1,020-bp DNA product from a wide range of monopartite and bipartite (A component only) begomoviruses, and C473 and PTYC1v2406, which preferentially amplifies a 859-bp DNA product from the monopartite TYLCV (1,2). Fragments of the expected size were obtained from all four samples, and all PCR products were sequenced. The sequences of the 1,020-bp PCR product from each of the four samples were compared and found to be 100% identical. The same was found for the 859-bp products. These sequences were compared with equivalent regions of begomoviruses and were identical to sequences of TYLCV. Since the two primer sets amplify overlapping regions of the TYLCV genome, the 1,020 and 859-bp products generated by the two primer sets from one plant were combined to create a 1,464-bp sequence that represented approximately half of the TYLCV genome and encompasses the C4 ORF, the intergenic region, and most of the coat protein gene. This 1,464-bp sequence from North Carolina was 99.2 to 99.6% identical to TYLCV sequences reported from Cuba (GenBank Accession No. AJ223505), the Dominican Republic (GenBank Accession No. AF024715), and Florida, and 96.9 to 98.2% identical to TYLCV sequences reported from the Bahamas, Israel (GenBank Accession No. X15656), Jamaica (GenBank Accession No. U84146), Mexico (GenBank Accession No. AF168709), and Spain (GenBank Accession No. AF071228). Symptomatic plants appeared to be infected with an isolate of TYLCV that is most similar to TYLCV isolates reported from Florida and the northeastern Caribbean. To our knowledge, this is the first report of TYLCV in North Carolina. TYLCV may have been introduced on transplants since the infected plants showed symptoms at an early growth stage. The appearance of infected plants in clusters of limited size suggests no spread or very limited spread in the field. Reports of populations of the whitefly (Bemisia tabaci) vector in the field were not available since whiteflies are not normally a problem in this area due to the higher altitude and relatively cool temperatures characteristic of Henderson County. It is not clear at this time what threat TYLCV poses to tomato production in the county, though its appearance indicates that the geographic range of TYLCV is continuing to expand in the southeastern United States. References: (1) M. Ghanim et al. Virology 240:295, 1998. (2) M. K. Nakhla et al. Phytopathol. Mediterr. 32:163, 1993. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993. (4) S. D. Wyatt et al. Phytopathology 86:1288, 1996.