A domino-like chlamydial attachment process: Parachlamydia acanthamoebae attachment to amoebae is concurrently required for several amoebal released molecules and serine-protease activity

Parachlamydia acanthamoebae is an obligate intracellular bacterium that infects free-living amoebae (Acanthamoeba), and is a potential human pathogen associated with hospital-acquired pneumonia. The attachment mechanism of this bacteria to host cells is a crucial step in bacterial pathogenesis, yet remains undetermined. Hence, we established monoclonal antibodies (mAbs) specific to either P. acanthamoebae or amoebae in an attempt to elucidate the involved attachment mechanism. Hybridomas of 954 clones were assessed, and we found four mAbs (mAb38, mAb300, mAb311, mAb562) that were reactive to the amoebae significantly inhibited bacterial attachment. All mAbs recognized amoebal released molecules, and mAb311 also recognized the amoebal surface. MAbs reacted with the bacteria not only in amoebae, but also those released from amoebae (except mAb311). Furthermore, serine-protease inhibitor had an inhibitory effect on the bacterial attachment to amoebae, although none of the mAbs had any synergetic effect on the attachment inhibition by the protease inhibitor. Taken together, we concluded that P. acanthoamebae attachment to amoebae is concurrently required for several amoebal released molecules and serine-protease activity, implying the existence of a complicated host-parasite relationship

Ciliates Expel Environmental Legionella-laden Pellets for Stockpiling Food

When the ciliate Tetrahymena is cultured with Legionella pneumophila, they expel bacteria packaged in free spherical pellets. Why the ciliates expel these pellets remains unclear. Hence, we determined optimal conditions for pellet expulsion, and assessed whether they contribute to maintenance of growth and survival of ciliates. When incubated environmental L. pneumophila, the ciliates maximally expelled the pellets at 2 days after infection. Heat-killed bacteria failed to produce pellets from ciliates, and there was no obvious difference in pellet production among the ciliates or bacterial strains. Morphological studies with assessment of lipid accumulation showed that pellets contained tightly packed bacteria with rapid lipid accumulation and were composed of the layers of membranes; bacterial culturability in the pellets rapidly decreased in contrast to that in ciliate-free culture, although the bacteria maintained membrane integrity in the pellets. Furthermore, ciliates newly cultured with pellets were maintained and grew vigorously compared with those without pellets. In contrast, a human L. pneumophila isolate killed ciliates 7 day post-infection in a Dot/Icm dependent manner and pellets harboring this strain did not support ciliate growth. Also, pellets harboring the human isolate were resuscitated by co-culture with amoebae, depending on Dot/Icm expression. Thus, while ciliates expel pellet-packaged environmental L. pneumophila for stockpiling food, the pellets packaged the human isolate are harmful on ciliate's survival, possibly connecting clinical significance

Leucine-rich α2-glycoprotein is a novel biomarker of neurodegenerative disease in human cerebrospinal fluid and causes neurodegeneration in mouse cerebral cortex.

Leucine-rich α2-glycoprotein (LRG) is a protein induced by inflammation. It contains a leucine-rich repeat (LRR) structure and easily binds with other molecules. However, the function of LRG in the brain during aging and neurodegenerative diseases has not been investigated. Here, we measured human LRG (hLRG) concentration in the cerebrospinal fluid (CSF) and observed hLRG expression in post-mortem human cerebral cortex. We then generated transgenic (Tg) mice that over-expressed mouse LRG (mLRG) in the brain to examine the effects of mLRG accumulation. Finally, we examined protein-protein interactions using a protein microarray method to screen proteins with a high affinity for hLRG. The CSF concentration of hLRG increases with age and is significantly higher in patients with Parkinson's disease with dementia (PDD) and progressive supranuclear palsy (PSP) than in healthy elderly people, idiopathic normal pressure hydrocephalus (iNPH) patients, and individuals with Alzheimer's disease (AD). Tg mice exhibited neuronal degeneration and neuronal decline. Accumulation of LRG in the brains of PDD and PSP patients is not a primary etiological factor, but it is thought to be one of the causes of neurodegeneration. It is anticipated that hLRG CSF levels will be a useful biomarker for the early diagnosis of PDD and PSP

Immunostaining results for LRG transgenic mice.

<p>(A) Immunostaining of the cerebral cortex of 4-, 8-, and 48-week old mice using NeuN antibody. 4-week-old WT mice, 4-week-old Tg mice, 8-week-old WT mice, 8-week-old Tg mice, 48-week-old WT mice, and 48-week-old Tg mice. Scale bar = 20µm. (B) A difference in the number of NeuN-positive cells was already observed between 4-week-old Tg and WT mice, but with 8- and 48-week old mice, there was a significant decline in the number of NeuN-positive cells in Tg mice compared to that in WT as determined by Mann-Whitney U test, **p < 0.001, Bars, <u>+</u> SD. (C) Cortical immunostaining for phosphorylated tau in 8-week-old WT, 8-week-old Tg, Tg mice neurons (lower panel, arrow), and Tg mice glial cells (lower panel, arrow head). Scale bar = 20µm (upper panel) and 10µm (lower panel). (D) Tg cerebral cortex demonstrated significantly more neurons and glial cells that were positive for phosphorylated tau than WT as determined by Mann-Whitney U test, **p < 0.001, Bars <u>+</u> SD. (E) NF-L immunostaining in the cortex of 8-week-old WT mice and the cortex of 8-week-old Tg mice. Large, winding dendritic neurons in Tg mice suggested that neurodegeneration had occurred. Scale bar = 20µm (upper panel) and 10µm (lower panel). WT = wild type, Tg = transgenic. </p

Human CSF analysis.

<p>Relationships between age and CSF total protein and LRG levels. (A) CSF total protein level did not correlate with age (r = 0.031, p = 0.731) (B) CSF LRG level significantly correlated with age (r = 0.314, p < 0.0001) using Spearman rank correlation. (C) CSF LRG level reverse correlated with the MMSE score (r = -0.271, p = 0.003) using Spearman rank correlation. LRG levels were significantly higher in the MMSE ≤ 23 group (113.6 ± 70.0 ng/ml) than in the MMSE > 23 group (92.0 ± 58.3 ng/ml), Mann-Whitney U test, p < 0.05). (D) CSF LRG levels tended to be higher in the iNPH groups (106.0 ± 46.7 ng/ml) vs. NC groups (44.0 ± 25.2 ng/ml), Mann-Whitney U test, p < 0.001. Concentrations of LRG in the PDD/DLB (251.5 ± 106.5 ng/ml) and PSP (261.1 ± 182.9 ng/ml) groups were significantly higher than those in the NC, iNPH, and AD groups (95.1 ± 64.4 ng/ml), Mann-Whitney U test, p < 0.001; PD/DLB vs. NC, p < 0.001; PSP vs. NC, p = 0.001; PDD/DLB vs. iNPH, p = 0.021, PSP vs. iNPH; p < 0.001, PDD/DLB vs. AD; p = 0.013, PSP vs. AD). ROC analysis of CSF biomarkers. LRG was the best discriminating biomarker for (E) PDD/DLB patients vs. NC, (F) PSP patients vs. NC, (G) iNPH patients vs. NC, (H) iNPH patients vs. PDD/DLB, and (I) iNPH patients vs. PSP, LRG was the most discriminating biomarker. CSF = cerebrospinal fluid, LRG = leucine-rich α2-glycoprotein. MMSE = Mini-Mental State Examination, iNPH = idiopathic normal pressure hydrocephalus, NC = normal control, PDD = Parkinson disease with dementia, DLB = dementia with Lewy bodies, PSP = Progressive Supranuclear Palsy, AD = Alzheimer disease, Amyloid(1-42) = amyloid beta peptide 1-42, p-TAU = phosphorylated tau, TAU = total tau.</p

LRG immunostaining of autopsied brains.

<p>(A) Western blotting of hLRG-transfected COS cell lysates showed strong expression of a 38-kDa band corresponding to LRG (arrow). <b>a</b>: COS cell lysate, <b>b</b>: hLRG-transfected COS cell lysates. (B) Immunoreactivity in hLRG-transfected cells was confirmed by immunostaining with anti-hLRG(329) rabbit IgG. COS cells, CHO-K1 cells, HEK293 cells. Scale bar = 20µm. (C) hLRG immunoreactivity in some glial cells and neurons in the internal pyramidal layer and multiform layer of the frontal cortex. Compared to the NC, LRG-immunoreactive cells were increased in iNPH. There was a significant increase in LRG-positive cells in PDD and PSP. With AD, we confirmed strong LRG immunoreactivity in neurons rather than in glial cells. Scale bar = 20µm (NC, iNPH, PDD, PSP and AD) and 10µm (Neuron and Glial cell). NC: 74-year-old male, frontal cortex iNPH: 76-year-old, male, frontal cortex. PDD: 69-year-old, male, frontal cortex. PSP: 65-year-old, male, temporal cortex, glial cell. AD: 82-year-old, male, frontal cortex, neuron. (D) Quantitation of hLRG immunoreactive cells in the cerebral cortex. Stained cells in a 0.05-mm² area of each cortical region were counted by independently by two investigators. The values on the y-axis represent the number of immunoreactive cells and are expressed as the means ± standard deviations for five areas in each case, and the results were analyzed using a one-way ANOVA followed by Dunnett’s test, *p < 0.05, **p < 0.001. Bars, <u>+</u> SD. </p