Live vaccines have long been known to trigger far more vigorous immune responses than their killed counterparts1–6. This has been attributed to the ability of live microorganisms to replicate and express specialized virulence factors that facilitate invasion and infection of their hosts7. However, protective immunization can often be achieved with a single injection of live, but not dead, attenuated microorganisms stripped of their virulence factors. Pathogen associated molecular patterns (PAMPs), which serve to alert the immune system8,9, are present in both live and killed vaccines, suggesting that certain poorly characterized aspects of live microorganisms, not incorporated in dead vaccines, are particularly effective at inducing protective immunity. Here we show that the innate immune system can directly sense microbial viability through detection of a special class of viability-associated PAMPs (vita-PAMPs). We identify prokaryotic messenger RNA (mRNA) as a vita-PAMP present only in viable bacteria, recognition of which elicits a unique innate response and a robust adaptive antibody response. Notably, the innate response evoked by viability and prokaryotic mRNA was thus far considered to be reserved for pathogenic bacteria, but we show that even nonpathogenic bacteria in sterile tissues can trigger similar responses, provided they are alive. Thus, the immune system actively gauges the infectious risk by scouring PAMPs for signatures of microbial life and thus infectivity. Detection of vita-PAMPs triggers an alert mode not warranted for dead bacteria. Vaccine formulations that incorporate vita-PAMPs could thus combine the superior protection of live vaccines with the safety of dead vaccines.

The outcome of cryptococcal pneumonia correlates with local macrophage polarization status, as M1 and M2 polarization marks protective and nonprotective responses, respectively. Overall, pulmonary macrophage polarization status changes over time during a cryptococcal infection. This could have been caused by repolarization of individual macrophages or by a replacement of M2-polarized cells by new M1-polarized cells. To explore the ability of macrophages to change between polarization states, we conducted a series of experiments using in vitro macrophages. Coculture of macrophages with Cryptococcus neoformans resulted in development of a weak M1-like phenotype, with modestly increased inducible nitric oxide synthase (iNOS) but lacking interleukin 6 (IL-6) induction. The C. neoformans-induced M1-like polarization state was plastic, as macrophages stimulated first with C. neoformans and then with gamma interferon (IFN-γ) or IL-4 expressed mRNA polarization patterns similar to those stimulated with cytokines alone. To further evaluate macrophage polarization plasticity, cytokine stimulatory conditions were established which fully polarized macrophages. IFN-γ and IL-4 stimulation differentially induced complete M1 and M2 polarization, defined by differential expression of marker mRNA panels, surface marker expression, and tumor necrosis factor alpha (TNF-α) protein production. Switching IFN-γ- to IL-4-stimulating conditions, and vice versa, resulted in uniform changes in profiles of polarization marker genes consistent with the most recent cytokine environment. Furthermore, the ability of sequentially stimulated macrophages to inhibit C. neoformans reflected the most recent polarizing condition, independent of previous polarization. Collectively, these data indicate that M1/M2 macrophage polarization phenotypes are highly plastic to external signals, and interventions which therapeutically repolarize macrophages could be beneficial for treatment of cryptococcosis.

A dissimilatory metal-and sulfur-reducing microorganism was isolated from surface sediments of a hydrocarbon-contaminated ditch in Norman, Okla. The isolate, which was designated strain PCA, was an obligately anaerobic, nonfermentative, nonmotile, gram-negative rod. PCA grew in a defined medium with acetate as an electron donor and ferric PP1, ferric oxyhydroxide, ferric citrate, elemental sulfur, Co(III)-EDTA, fumarate, or malate as the sole electron acceptor. PCA also coupled the oxidation of hydrogen to the reduction of Fe(III) but did not reduce Fe(III) with sulfur, glucose, lactate, fumarate, propionate, butyrate, isobutryate, isovalerate, succinate, yeast extract, phenol, benzoate, ethanol, propanol, or butanol as an electron donor. PCA did not reduce oxygen, Mn(IV), U(VI), nitrate, sulfate, sulfite, or thiosulfate with acetate as the electron donor. Cell suspensions of PCA exhibited dithionite-reduced minus air-oxidized difference spectra which were characteristic of c-type cytochromes. Phylogenetic analysis of the 16S rRNA sequence placed PCA in the delta subgroup of the proteobacteria. Its closest known relative is Geobacter metalireducens. The ability to utilize either hydrogen or acetate as the sole electron donor for Fe(III) reduction makes strain PCA a unique addition to the relatively small group of respiratory metal-reducing microorganisms available in pure culture. A new species name, Geobacter su(furreducens, is proposed.

Rats were given 10 light-shock pairings on 2 successive days. At 24-48 hr following training, groups of rats received bilateral transection of the cerebellar peduncles, bilateral lesions of the red nucleus (which receives most of the cerebellar efferents), or bilateral lesions of the central nucleus of the amygdala. Control rats were sham operated. At 3-4 days after surgery, the rats were tested for potentiated startle (increased acoustic startle in the presence of the light previously paired with shock). Potentiated startle was blocked by lesions of the central nucleus of the amygdala. Transection of the cerebellar peduncles or lesions of the red nucleus did not block potentiated startle. A second experiment in which a visual prepulse test was used indicated that the blockade of potentiated startle observed in the animals with amygdala lesions could not be attributed to optic tract damage. A third experiment demonstrated that the absence of potentiation in the animals with amygdala lesions was not simply due to a lowered startle level ceiling, because these animals could show increased startle with increased stimulus intensity and with administration of strychnine, a drug that increases startle. Taken together, the results are consistent with the hypothesis that the amygdala is involved in fear conditioning, because potentiated startle is a measure of conditioned fear.

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In Experiment 1 four groups of rats received 30 light-shock pairings using footshock intensities of either .2, .4, .8, or 1.6 mA. One day later all rats were tested for startle by presenting tones in the presence or absence of the light CS. Potentiated startle (the difference between startle on light-tone vs tone-alone trials) was nonmonotonically related to the shock intensity used in training, with the greatest potentiation at intermediate shock levels. Experiment 3 demonstrated a similar relationship when backshocks instead of footshocks were used. In Experiment 2 rats were trained with either a moderate or high shock and then given an extended extinction-test session 1 day later. The moderate-shock group showed a gradual decline in potentiated startle over extinction. The high-shock group showed a nonmonotonic extinction curve where potentiation progressively increased toward the middle of extinction and dissipated thereafter. The results suggest that acoustic startle bears an inverted U-shaped relationship to fear and are discussed in relation to other studies concerned with this issue.

Huanglongbing (HLB), also known as citrus greening, is a lethal disease of citrus caused by several species of 'Candidatus Liberibacter', a psyllid-transmitted, phloem-limited, alpha proteobacteria. 'Ca. Liberibacter asiaticus' is widespread in Florida citrus. The recently published 'Ca. L. asiaticus' psy62 genome, derived from a psyllid, revealed a prophage-like region of DNA in the genome, but phage have not been associated with 'Ca. L. asiaticus' to date. In the present study, shotgun sequencing and a fosmid DNA library of curated 'Ca. L. asiaticus' UF506, originally derived from citrus symptomatic for HLB, revealed two largely homologous, circular phage genomes, SC1 and SC2. SC2 encoded putative adhesin and peroxidase genes that had not previously been identified in 'Ca. L. asiaticus' and which may be involved in lysogenic conversion. SC2 also appeared to lack lytic cycle genes and replicated as a prophage excision plasmid, in addition to being found integrated in tandem with SC1 in the UF506 chromosome. By contrast, SC1 carried suspected lytic cycle genes and was found in nonintegrated, lytic cycle forms only in planta. Phage particles associated with 'Ca. L. asiaticus' were found in the phloem of infected periwinkles by transmission electron microscopy. In psyllids, both SC1 and SC2 were found only as prophage.